WO2020007702A1

WO2020007702A1 - Antisense oligonucleotides targeting bcl2l11

Info

Publication number: WO2020007702A1
Application number: PCT/EP2019/067145
Authority: WO
Inventors: Eva Marie Lindholm; Steffen Schmidt
Original assignee: Roche Innovation Center Copenhagen AS
Current assignee: Roche Innovation Center Copenhagen AS
Priority date: 2018-07-02
Filing date: 2019-06-27
Publication date: 2020-01-09
Anticipated expiration: 2021-01-02

Abstract

The present invention relates to antisense LNA oligonucleotides (oligomers) complementary to BCL2L11 pre-mRNA intron and exon sequences, which are capable of inhibiting the expression of BCL2L11 protein. Inhibition of BCL2L11 expression is beneficial for a range of medical disorders including hepatotoxicity, Alzheimer´s disease, stroke, epileptic seizures, encephalomyelitis, multiple sclerosis (MS), type 1 diabetes, type 2 diabetes, and graft- versus-host disease.

Description

ANTISENSE OLIGONUCLEOTIDES TARGETING BCL2L11

FIELD OF INVENTION

The present invention relates to antisense LNA oligonucleotides (oligomers) complementary to BCL2L11 pre-mRNA exon sequences, which are capable of inhibiting the expression of BCL2L1 1. Inhibition of BCL2L11 expression is beneficial for a range of medical disorders including hepatotoxicity, Alzheimer^'s disease, stroke, epileptic seizures, encephalomyelitis, multiple sclerosis (MS), type 1 diabetes, type 2 diabetes, and graft-versus-host disease.

BACKGROUND

BCL2L11 (B-cell-lymphoma 2-like 11 or BCL2-like 1 1 ) frequently also referred to as BIM (BCL-2 interacting mediator of cell death) is a protein belonging to the BCL-2 family.

Members of this family form hetero- and homodimers and are important regulators of programmed cell death via the intrinsic apoptotic pathway. BCL2L1 1 has been implicated to play a role in diseases associated with apoptosis, autophagy and inflammation (O'Connor et al„ EMBO, 2012, 17(2): 384-395).

The Bcl2L11 gene has 6 exons and undergoes alternative splicing to form at least 18 different isoforms transcribed from mRNA harboring 3-6 exons of which 2-4 are coding exons (Sionov et al., Oncotarget. 2015 Sep 15; 6(27): 23058-23134).

Overexpression of BCL2L11 has been shown to be associated with a favorable prognosis for cancer patients (Kim et al., Anticancer Res. 2017 Sep;37(9):4873-4879). Also, decreased expression of BCL2L11 is thought to mediate anti-cancer drug-resistance (Tsubaki et al., Leuk Res. 2012 Oct;36(10): 1315-22). It has been suggested that drug-resistant cancer cells can be resensitized to anti-cancer drugs by using splice-switching antisense oligonucleotides which block exon 3 of the BCL2L11 gene, but enhance exon 4 splicing (Liu et al.,

Oncotarget. 2017; 8(44):77567-77585).

Expression of the BCL2L11 gene is induced by nerve growth factor (NGF) and the forkhead transcription factor FKHR-L1. Reducing BCL2L11 expression by antisense oligonucleotides was shown to promote neuronal survival after NGF withdrawal (Whitfield et al., Neuron. 2001 Mar; 29(3):629-43). Since down-regulation of BCL2L11 has neuroprotective effects, it has been suggested that therapeutics which target BCL2L11 may reduce cell damage following neurological insults (Meller et al., The Journal of biological chemistry. 2006;281 (11 ):7429- 7436; Murphy et al., Cell Death and Differentiation, 2010, 17(3), 459-468). Further, it has been shown that BCL2L11 expression is significantly elevated in a vulnerable population of neurons in Alzheimer’s disease. Therefore, the BCL2L11 gene is considered as target for therapeutic intervention in Alzheimer's disease as (Biswas et al., Journal of Neuroscience, 2007, 27 (4) 893-900).

Moreover, administration of potentially hepatotoxic high affinity gapmer antisense oligonucleotides containing locked nucleic acid substitutions resulted in upregulation of BCL2L11 expression (Dieckmann et al., Molecular Therapy Nucleic Acids. 2018; 10: 45-54).

In another study, it was shown that BCL2L22-deficient mice were substantially protected from APAP-induced liver damage.

BCL2L1 1 plays also a role in autoimmunity, in particular autoimmune encephalomyelitis and diabetes. Deletion of BCL2L11 in hematopoietic cells rendered mice resistant to autoimmune encephalomyelitis and diabetes (Ludwinski et al., The Journal of clinical investigation, 2009,

1 19(6):1706-1713). Further, inhibiting the expression of BCL2L11 may also prevent allograft rejection (Yu, et al., Am J Blood Res. 2012; 2(1 ): 77-85).

WO2011/146674 discloses antisense oligonucleotides that target natural occurring antisense polynucleotides of the BCL2L11 gene. The administration of the antisense oligonucleotides resulted in upregulation of BCL2L11 expression.

There is a need for therapeutic agents which can inhibit BCL2L11 specifically.

We have screened a large number of LNA gapmers targeting mouse and human BCL2L11 and identified sequences and compounds which are particularly potent and effective to specifically target for BCL2L1 1 antisense in vitro (human and mouse cells).

OBJECTIVE OF THE INVENTION

The inventors have identified particularly effective regions of the BCL2L1 1 transcript (BCL2L11) for antisense inhibition in vitro or in vivo, and provides for antisense

oligonucleotides, including LNA gapmer oligonucleotides, which target these regions of the BCL2L11 premRNA or mature mRNA. The present invention identifies oligonucleotides which inhibit human BCL2L11 which are useful in the treatment of a range of medical disorders including hepatotoxicity, Alzheimer^'s disease, stroke, epileptic seizures, encephalomyelitis, multiple sclerosis (MS), type 1 diabetes, type 2 diabetes, and graft- versus-host disease.

STATEMENT OF THE INVENTION The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human BCL2L11 target nucleic acid, wherein the antisense oligonucleotide is capable of inhibiting the expression of human BCL2L1 1 in a cell which is expressing human BCL2L1 1.

The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human BCL2L11 target nucleic acid, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully

complementary, to a sequence selected from the group consisting of SEQ ID NO 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32 and 33.

complementary to SEQ ID NO 15.

The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90%

complementary, such as fully complementary, to SEQ ID NO 15 wherein the antisense oligonucleotide is capable of inhibiting the expression of human BCL2L1 1 in a cell which is expressing human BCL2L11.

The invention provides for an LNA antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90%

complementary, such as fully complementary, to SEQ ID NO 15, wherein the antisense oligonucleotide is capable of inhibiting the expression of human BCL2L1 1 in a cell which is expressing human BCL2L11.

The invention provides for a gapmer antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to SEQ ID NO 15 wherein the antisense oligonucleotide is capable of inhibiting the expression of human BCL2L1 1 in a cell which is expressing human BCL2L11.

The invention provides for an LNA gapmer antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to SEQ ID NO 15 wherein the antisense oligonucleotide is capable of inhibiting the expression of human BCL2L1 1 in a cell which is expressing human BCL2L11.

The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to a sequence selected from the group consisting of SEQ ID NO 11 , SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 14, wherein the antisense oligonucleotide is capable of inhibiting the expression of human BCL2L11 in a cell which is expressing human BCL2L11.

The invention provides for an LNA antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to a sequence selected from the group consisting of SEQ ID NO 11 , SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 14, wherein the antisense oligonucleotide is capable of inhibiting the expression of human BCL2L11 in a cell which is expressing human BCL2L11.

The invention provides for a gapmer antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to a sequence selected from the group consisting of SEQ ID NO 11 , SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 14, wherein the antisense oligonucleotide is capable of inhibiting the expression of human BCL2L11 in a cell which is expressing human BCL2L11. The invention provides for an LNA gapmer antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to a sequence selected from the group consisting of SEQ ID NO 11 , SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 14, wherein the antisense oligonucleotide is capable of inhibiting the expression of human BCL2L11 in a cell which is expressing human BCL2L11.

The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to SEQ ID NO 1 1 wherein the antisense oligonucleotide is capable of inhibiting the expression of human BCL2L11 transcript in a cell which is expressing human BCL2L11 transcript.

The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to SEQ ID NO 12 wherein the antisense oligonucleotide is capable of inhibiting the expression of human BCL2L11 transcript in a cell which is expressing human BCL2L11 transcript.

The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to SEQ ID NO 13 wherein the antisense oligonucleotide is capable of inhibiting the expression of human BCL2L11 transcript in a cell which is expressing human BCL2L11 transcript.

The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to SEQ ID NO 14 wherein the antisense oligonucleotide is capable of inhibiting the expression of human BCL2L11 transcript in a cell which is expressing human BCL2L11 transcript.

The oligonucleotide of the invention as referred to or claimed herein may be in the form of a pharmaceutically acceptable salt.

The invention provides for a conjugate comprising the oligonucleotide according to the invention, and at least one conjugate moiety covalently attached to said oligonucleotide.

The invention provides for a pharmaceutical composition comprising the oligonucleotide or conjugate of the invention and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

The invention provides for an in vivo or in vitro method for modulating BCL2L11 expression in a target cell which is expressing BCL2L11, said method comprising administering an oligonucleotide or conjugate or pharmaceutical composition of the invention in an effective amount to said cell.

The invention provides for a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide, conjugate or the pharmaceutical composition of the invention to a subject suffering from or susceptible to the disease.

In some embodiments, the disease is selected from the group consisting of hepatotoxicity, Alzheimer^'s disease, stroke, epileptic seizures, encephalomyelitis, multiple sclerosis (MS), type 1 diabetes, type 2 diabetes, and graft-vers us-host disease.

The invention provides for the oligonucleotide, conjugate or the pharmaceutical composition of the invention for use in medicine.

The invention provides for the oligonucleotide, conjugate or the pharmaceutical composition of the invention for use in the treatment or prevention of a disease selected from the group consisting of hepatotoxicity, Alzheimer^'s disease, stroke, epileptic seizures,

encephalomyelitis, multiple sclerosis (MS), type 1 diabetes, type 2 diabetes, and graft- versus-host disease.

The invention provides for the use of the oligonucleotide, conjugate or the pharmaceutical composition of the invention, for the preparation of a medicament for treatment or prevention of a disease selected from the group consisting of hepatotoxicity, Alzheimer^'s disease, stroke, epileptic seizures, encephalomyelitis, multiple sclerosis (MS), type 1 diabetes, type 2 diabetes, and graft-versus-host disease. BRIEF DESCRIPTION OF FIGURES

Figure 1 : Testing in vitro efficacy of various antisense oligonucleotides targeting human BCL2L11 mRNA in A549 and THP-1 cell lines at single concentration.

Figure 2: Testing in vitro efficacy of various antisense oligonucleotides targeting mouse Bcl2l11 mRNA in J774A.1 and MPC-1 1 cell lines at single concentration.

Figure 3: Comparison of in vitro efficacy for antisense oligonucleotides targeting human BCL2L11 mRNA in A549 and THP-1 cell lines at single concentration shows good correlation. Two motifs with very efficient targeting are highlighted.

Figure 4: Comparison of in vitro efficacy for antisense oligonucleotides targeting mouse Bcl2l11 mRNA in J774A.1 and MPC-1 1 cell lines at single concentration shows good correlation. Two motifs with very efficient targeting are highlighted.

Figure 5: IC50 values for selected oligonucleotides targeting human BCL2L11 mRNA in vitro in A549 and THP-1 cell lines.

Figure 6: Testing selected oligonucleotides targeting human BCL2L11 mRNA in vitro for concentration dependent potency and efficacy in THP-1 cell line.

Figure 7: Testing selected oligonucleotides targeting human BCL2L11 mRNA in vitro for concentration dependent potency and efficacy in A431 cell line.

Figure 8: IC50 values for selected oligonucleotides targeting mouse Bcl2l11 mRNA in vitro in J774A.1 and MPC-1 1 cell lines.

Figure 9: Testing selected oligonucleotides targeting mouse Bcl2l11 mRNA in vitro for concentration dependent potency and efficacy in J774A.1 cell line.

Figure 10: Testing selected oligonucleotides targeting mouse Bcl2l11 mRNA in vitro for concentration dependent potency and efficacy in MPC-1 1 cell line.

Figure 11 : Mouse in vivo efficacy: remaining Bcl2H 1 transcript in mouse tissues after 7 days of treatment, Intravenous IV (tail vein).

DEFINITIONS

Oligonucleotide

The term“oligonucleotide” as used herein is defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are commonly made in the laboratory by solid-phase chemical synthesis followed by purification. When referring to a sequence of the oligonucleotide, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides. The oligonucleotide of the invention is man-made, and is chemically synthesized, and is typically purified or isolated. The oligonucleotide of the invention may comprise one or more modified nucleosides or nucleotides.

Antisense oligonucleotides

The term“Antisense oligonucleotide” as used herein is defined as oligonucleotides capable of modulating expression of a target gene by hybridizing to a target nucleic acid, in particular to a contiguous sequence on a target nucleic acid. The antisense oligonucleotides are not essentially double stranded and are therefore not siRNAs or shRNAs. Preferably, the antisense oligonucleotides of the present invention are single stranded. It is understood that single stranded oligonucleotides of the present invention can form hairpins or intermolecular duplex structures (duplex between two molecules of the same oligonucleotide), as long as the degree of intra or inter self-complementarity is less than 50% across of the full length of the oligonucleotide

Contiguous Nucleotide Sequence

The term“contiguous nucleotide sequence” refers to the region of the oligonucleotide which is complementary to the target nucleic acid. The term is used interchangeably herein with the term“contiguous nucleobase sequence” and the term“oligonucleotide motif sequence”.

In some embodiments all the nucleotides of the oligonucleotide constitute the contiguous nucleotide sequence. In some embodiments the oligonucleotide comprises the contiguous nucleotide sequence, such as a F-G-F’ gapmer region, and may optionally comprise further nucleotide(s), for example a nucleotide linker region which may be used to attach a functional group to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid. Adventurously, the contiguous nucleotide sequence is 100% complementary to the target nucleic acid.

Nucleotides

Nucleotides are the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention include both naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides, such as DNA and RNA nucleotides comprise a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (which is absent in nucleosides). Nucleosides and nucleotides may also interchangeably be referred to as“units” or“monomers”.

Modified nucleoside

The term“modified nucleoside” or“nucleoside modification” as used herein refers to nucleosides modified as compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the (nucleo)base moiety. In a preferred embodiment the modified nucleoside comprise a modified sugar moiety. The term modified nucleoside may also be used herein interchangeably with the term“nucleoside analogue” or modified“units” or modified“monomers”. Nucleosides with an unmodified DNA or RNA sugar moiety are termed DNA or RNA nucleosides herein. Nucleosides with modifications in the base region of the DNA or RNA nucleoside are still generally termed DNA or RNA if they allow Watson Crick base pairing.

Modified internucleoside linkages

The term“modified internucleoside linkage” is defined as generally understood by the skilled person as linkages other than phosphodiester (PO) linkages, that covalently couples two nucleosides together. The oligonucleotides of the invention may therefore comprise modified internucleoside linkages. In some embodiments, the modified internucleoside linkage increases the nuclease resistance of the oligonucleotide compared to a phosphodiester linkage. For naturally occurring oligonucleotides, the internucleoside linkage includes phosphate groups creating a phosphodiester bond between adjacent nucleosides. Modified internucleoside linkages are particularly useful in stabilizing oligonucleotides for in vivo use, and may serve to protect against nuclease cleavage at regions of DNA or RNA nucleosides in the oligonucleotide of the invention, for example within the gap region of a gapmer oligonucleotide, as well as in regions of modified nucleosides, such as region F and F’.

In an embodiment, the oligonucleotide comprises one or more internucleoside linkages modified from the natural phosphodiester, such one or more modified internucleoside linkages that is for example more resistant to nuclease attack. Nuclease resistance may be determined by incubating the oligonucleotide in blood serum or by using a nuclease resistance assay (e.g. snake venom phosphodiesterase (SVPD)), both are well known in the art. Internucleoside linkages which are capable of enhancing the nuclease resistance of an oligonucleotide are referred to as nuclease resistant internucleoside linkages. In some embodiments at least 50% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are modified, such as at least 60%, such as at least 70%, such as at least 80 or such as at least 90% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are nuclease resistant internucleoside linkages. In some embodiments all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are nuclease resistant internucleoside linkages. It will be recognized that, in some embodiments the nucleosides which link the oligonucleotide of the invention to a non-nucleotide functional group, such as a conjugate, may be phosphodiester. A preferred modified internucleoside linkage is phosphorothioate.

Phosphorothioate internucleoside linkages are particularly useful due to nuclease resistance, beneficial pharmacokinetics and ease of manufacture. In some embodiments at least 50% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate, such as at least 60%, such as at least 70%, such as at least 80% or such as at least 90% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate. In some embodiments all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate.

Nuclease resistant linkages, such as phosphorothioate linkages, are particularly useful in oligonucleotide regions capable of recruiting nuclease when forming a duplex with the target nucleic acid, such as region G for gapmers. Phosphorothioate linkages may, however, also be useful in non-nuclease recruiting regions and/or affinity enhancing regions such as regions F and F’ for gapmers. Gapmer oligonucleotides may, in some embodiments comprise one or more phosphodiester linkages in region F or F’, or both region F and F’, which the internucleoside linkage in region G may be fully phosphorothioate.

Advantageously, all the internucleoside linkages in the contiguous nucleotide sequence of the oligonucleotide are phosphorothioate linkages.

It is recognized that, as disclosed in EP2 742 135, antisense oligonucleotide may comprise other internucleoside linkages (other than phosphodiester and phosphorothioate), for example alkyl phosphonate / methyl phosphonate internucleosides, which according to EP2 742 135 may for example be tolerated in an otherwise DNA phosphorothioate gap region.

Nucleobase

The term nucleobase includes the purine (e.g. adenine and guanine) and pyrimidine (e.g. uracil, thymine and cytosine) moiety present in nucleosides and nucleotides which form hydrogen bonds in nucleic acid hybridization. In the context of the present invention the term nucleobase also encompasses modified nucleobases which may differ from naturally occurring nucleobases, but are functional during nucleic acid hybridization. In this context “nucleobase” refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, as well as non-naturally occurring variants. Such variants are for example described in Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid

Chemistry Suppl. 37 1.4.1.

In a some embodiments the nucleobase moiety is modified by changing the purine or pyrimidine into a modified purine or pyrimidine, such as substituted purine or substituted pyrimidine, such as a nucleobased selected from isocytosine, pseudoisocytosine, 5-methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5- thiazolo-uracil, 2-thio-uracil, 2’thio-thymine, inosine, diaminopurine, 6-aminopurine, 2- aminopurine, 2,6-diaminopurine and 2-chloro-6-aminopurine.

The nucleobase moieties may be indicated by the letter code for each corresponding nucleobase, e.g. A, T, G, C or U, wherein each letter may optionally include modified nucleobases of equivalent function. For example, in the exemplified oligonucleotides, the nucleobase moieties are selected from A, T, G, C, and 5-methyl cytosine. Optionally, for LNA gapmers, 5-methyl cytosine LNA nucleosides may be used.

Modified oligonucleotide

The term modified oligonucleotide describes an oligonucleotide comprising one or more sugar-modified nucleosides and/or modified internucleoside linkages. The term chimeric” oligonucleotide is a term that has been used in the literature to describe oligonucleotides with modified nucleosides.

Complementarity

The term“complementarity” describes the capacity for Watson-Crick base-pairing of nucleosides/nucleotides. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A) - thymine (T)/uracil (U). It will be understood that oligonucleotides may comprise nucleosides with modified nucleobases, for example 5-methyl cytosine is often used in place of cytosine, and as such the term complementarity encompasses Watson Crick base-paring between non-modified and modified nucleobases (see for example Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009)

Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1 ).

The term“% complementary” as used herein, refers to the number of nucleotides in percent of a contiguous nucleotide sequence in a nucleic acid molecule (e.g. oligonucleotide) which, at a given position, are complementary to ( i.e . form Watson Crick base pairs with) a contiguous sequence of nucleotides, at a given position of a separate nucleic acid molecule (e.g. the target nucleic acid or target sequence). The percentage is calculated by counting the number of aligned bases that form pairs between the two sequences (when aligned with the target sequence 5’-3’ and the oligonucleotide sequence from 3’-5’), dividing by the total number of nucleotides in the oligonucleotide and multiplying by 100. In such a comparison a nucleobase/nucleotide which does not align (form a base pair) is termed a mismatch.

Preferably, insertions and deletions are not allowed in the calculation of % complementarity of a contiguous nucleotide sequence.

The term“fully complementary”, refers to 100% complementarity. Identity

The term“Identity” as used herein, refers to the proportion of nucleotides (expressed in percent) of a contiguous nucleotide sequence in a nucleic acid molecule (e.g.

oligonucleotide) which across the contiguous nucleotide sequence, are identical to a reference sequence (e.g. a sequence motif). The percentage of identity is thus calculated by counting the number of aligned bases that are identical (a match) between two sequences (e.g. in the contiguous nucleotide sequence of the compound of the invention and in the reference sequence), dividing that number by the total number of nucleotides in the aligned region and multiplying by 100. Therefore, Percentage of Identity = (Matches x 100)/Length of aligned region (e.g. the contiguous nucleotide sequence). Insertions and deletions are not allowed in the calculation the percentage of identity of a contiguous nucleotide sequence. It will be understood that in determining identity, chemical modifications of the nucleobases are disregarded as long as the functional capacity of the nucleobase to form Watson Crick base pairing is retained (e.g. 5-methyl cytosine is considered identical to a cytosine for the purpose of calculating % identity).

Hybridization

The term“hybridizing” or“hybridizes” as used herein is to be understood as two nucleic acid strands (e.g. an oligonucleotide and a target nucleic acid) forming hydrogen bonds between base pairs on opposite strands thereby forming a duplex. The affinity of the binding between two nucleic acid strands is the strength of the hybridization. It is often described in terms of the melting temperature (T_m) defined as the temperature at which half of the oligonucleotides are duplexed with the target nucleic acid. At physiological conditions T_m is not strictly proportional to the affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard state Gibbs free energy AG° is a more accurate representation of binding affinity and is related to the dissociation constant (K_d) of the reaction by AG°=-RTIn(K_d), where R is the gas constant and T is the absolute temperature. Therefore, a very low AG° of the reaction between an oligonucleotide and the target nucleic acid reflects a strong

hybridization between the oligonucleotide and target nucleic acid. AG° is the energy associated with a reaction where aqueous concentrations are 1 M, the pH is 7, and the temperature is 37°C. The hybridization of oligonucleotides to a target nucleic acid is a spontaneous reaction and for spontaneous reactions AG° is less than zero. AG° can be measured experimentally, for example, by use of the isothermal titration calorimetry (ITC) method as described in Hansen et al., 1965, Chem. Comm. 36-38 and Holdgate et a!., 2005, Drug Discov Today. The skilled person will know that commercial equipment is available for DQ° measurements. DQ° can also be estimated numerically by using the nearest neighbor model as described by SantaLucia, 1998, Proc Natl Acad Sci USA. 95: 1460-1465 using appropriately derived thermodynamic parameters described by Sugimoto et al., 1995, Biochemistry 34:11211-1 1216 and McTigue et al., 2004, Biochemistry 43:5388-5405. In order to have the possibility of modulating its intended nucleic acid target by hybridization, oligonucleotides of the present invention hybridize to a target nucleic acid with estimated DQ° values below -10 kcal for oligonucleotides that are 10-30 nucleotides in length. In some embodiments the degree or strength of hybridization is measured by the standard state Gibbs free energy DQ°. The oligonucleotides may hybridize to a target nucleic acid with estimated DQ° values below the range of -10 kcal, such as below -15 kcal, such as below - 20 kcal and such as below -25 kcal for oligonucleotides that are 8-30 nucleotides in length.

In some embodiments the oligonucleotides hybridize to a target nucleic acid with an estimated DQ° value of -10 to -60 kcal, such as -12 to -40, such as from -15 to -30 kcal or- 16 to -27 kcal such as -18 to -25 kcal.

Target nucleic acid

According to the present invention, the target nucleic acid is a nucleic acid which encodes mammalian BCL2L1 1 and may for example be a gene, a BCL2L11 RNA, a mRNA, a pre- mRNA, a mature mRNA or a cDNA sequence. The target may therefore be referred to as an BCL2L1 1 target nucleic acid.

Suitably, the target nucleic acid encodes an BCL2L11 protein, in particular mammalian BCL2L1 1 , such as the human BCL2L1 1 gene encoding pre-mRNA or mRNA sequences provided herein as SEQ ID NO 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32 or 33.

In some embodiments, the target nucleic acid is selected from the group consisting of SEQ ID NO 15 or 16 or naturally occurring variants thereof (e.g. BCL2L11 sequences encoding a mammalian BCL2L1 1 protein).

If employing the oligonucleotide of the invention in research or diagnostics the target nucleic acid may be a cDNA or a synthetic nucleic acid derived from DNA or RNA.

For in vivo or in vitro application, the oligonucleotide of the invention is typically capable of inhibiting the expression of the BCL2L11 target nucleic acid in a cell which is expressing the BCL2L11 target nucleic acid. The contiguous sequence of nucleobases of the

oligonucleotide of the invention is typically complementary to the BCL2L11 target nucleic acid, as measured across the length of the oligonucleotide, optionally with the exception of one or two mismatches, and optionally excluding nucleotide based linker regions which may link the oligonucleotide to an optional functional group such as a conjugate, or other non- complementary terminal nucleotides (e.g. region D’ or D”). The target nucleic acid is a messenger RNA, such as a mature mRNA or a pre-mRNA which encodes mammalian BCL2L1 1 protein, such as human BCL2L1 1 , e.g. the human BCL2L11 pre-mRNA sequence, such as that disclosed as SEQ ID NO 15, or BCL2L11 mature mRNA, such as that disclosed as SEQ ID NO 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32 or 33. SEQ ID NOs 15 - 33 are DNA sequences - it will be understood that target RNA sequences have uracil (U) bases in place of the thymidine bases (T).

n some embodiments, the oligonucleotide of the invention targets SEQ ID NO 15.

n some embodiments, the oligonucleotide of the invention targets SEQ ID NO 16.

n some embodiments, the oligonucleotide of the invention targets SEQ ID NO 17.

n some embodiments, the oligonucleotide of the invention targets SEQ ID NO 18.

n some embodiments, the oligonucleotide of the invention targets SEQ ID NO 19.

n some embodiments, the oligonucleotide of the invention targets SEQ ID NO 20.

n some embodiments, the oligonucleotide of the invention targets SEQ ID NO 21.

n some embodiments, the oligonucleotide of the invention targets SEQ ID NO 22.

n some embodiments, the oligonucleotide of the invention targets SEQ ID NO 23.

n some embodiments, the oligonucleotide of the invention targets SEQ ID NO 24.

n some embodiments, the oligonucleotide of the invention targets SEQ ID NO 25.

n some embodiments, the oligonucleotide of the invention targets SEQ ID NO 26.

n some embodiments, the oligonucleotide of the invention targets SEQ ID NO 27.

n some embodiments, the oligonucleotide of the invention targets SEQ ID NO 28.

n some embodiments, the oligonucleotide of the invention targets SEQ ID NO 29.

n some embodiments, the oligonucleotide of the invention targets SEQ ID NO 30.

n some embodiments, the oligonucleotide of the invention targets SEQ ID NO 31.

n some embodiments, the oligonucleotide of the invention targets SEQ ID NO 32.

n some embodiments, the oligonucleotide of the invention targets SEQ ID NO 33.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 15 and at least one of, such as two, three, four, five, six, seven, eight, nine, ten or more of SEQ ID NO 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32 and 33.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32 and 33.

In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 15, 16, 17, 18, 20, 23, 25, 26, 27, 28, 29, 30, 31 , 32.

Target Sequence

The term“target sequence” as used herein refers to a sequence of nucleotides present in the target nucleic acid which comprises the nucleobase sequence which is complementary to the oligonucleotide of the invention. In some embodiments, the target sequence consists of a region on the target nucleic acid which is complementary to the contiguous nucleotide sequence of the oligonucleotide of the invention.

Herein are provided numerous target sequence regions, as defined by regions of the human BCL2L1 1 pre-mRNA (using SEQ ID NO 15 as a reference) which may be targeted by the oligonucleotides of the invention.

In some embodiments the target sequence is longer than the complementary sequence of a single oligonucleotide, and may, for example represent a preferred region of the target nucleic acid which may be targeted by several oligonucleotides of the invention.

The oligonucleotide of the invention comprises a contiguous nucleotide sequence which is complementary to or hybridizes to the target nucleic acid, such as a sub-sequence of the target nucleic acid, such as a target sequence described herein.

The oligonucleotide comprises a contiguous nucleotide sequence which are complementary to a target sequence present in the target nucleic acid molecule. The contiguous nucleotide sequence (and therefore the target sequence) comprises of at least 10 contiguous nucleotides, such as 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleotides, such as from 12-25, such as from 14-18 contiguous nucleotides.

Target Sequence Regions

The inventors have identified particularly effective sequences of the BCL2L11 target nucleic acid which may be targeted by the oligonucleotide of the invention.

In some embodiments the target sequence is SEQ ID NO 1 1 .

In some embodiments the target sequence is SEQ ID NO 12.

In some embodiments the target sequence is SEQ ID NO 13.

In some embodiments the target sequence is SEQ ID NO 14.

SEQ ID NO 1 1 : AGCAACCTT CT GAT GT AAGTT CT GAGT GT G (15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33)

SEQ ID NO 12: TAG G AC C C AG C GTATGT AG CATTT GT ATT G (15, 16, 17, 18, 20, 23, 25, 26, 27, 28, 29, 30, 31 , 32)

SEQ ID NO 13: CTT CT GAT GT AAGTT CT GAG (15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33) SEQ ID NO 14: C C C AG CGTATGTAG C ATTT G (15, 16, 17, 18, 20, 23, 25, 26, 27, 28, 29, 30, 31, 32)

(numbers in brackets refer to the SEQ ID of BCL2L11 premRNA or mRNA transcripts in which the target sequence is found).

In some embodiments the oligonucleotide of the invention, or contiguous nucleotide sequence thereof is complementary, such as fully complementary, to a region of SEQ ID NO 15, selected from the group consisting of 306 - 320; 3219 - 3239; 3228 - 3250; 3252 - 3331;

3333 - 3352 3354 - 3384 3441 - 3465 3524 - 3557; 3560 - 3596 3606 - 3624 3626 - 3640 3713 -3736 3751 - 3773 3777 - 3868 3870 - 3955; 3957 - 3971 3995 - 4029 4043 - 4062 4064 -4081 4103-4162 4165-4182 4184-4227; 4229-4243 4243 - 4267 4310-4342 4344 - 4499 4501 - 4568 4570-4819 4821 - 4835; 4837 - 4852 4880-4910 4916-4933 4938 -4971 4973 - 4989 4996 - 5023 5039 -5086; 5088 -5113 5130-5156 5158-5193 5218-5279 5309 -5361 5367 - 5426 5430 - 5448; 5456 - 5501 5523 - 5546 5548 - 5565 5567 - 5594 5596-5641 5643 - 5666 5668 - 5682; 5684 - 5720 5722 - 5740 5742 - 5764 5761 -5783 5785-5831 5837 - 5886 5903 - 5920; 5922 - 5937 5949 - 5965 5978-6010 6012 -6031 6037 - 6051 6059 - 6074 6081 -6097; 6099 -6117 6133-6150 6169-6204 6215-6239 6263 - 6320 6342 - 6362 6379 -6397; 6410-6426 6428 - 6457 6459 - 6474 6476 - 6492 6509 - 6535 6548 - 6583 6585 - 6604; 6640 - 6688 6690 - 6708 6710-6746 6753 -6811 6813 -6836 6853 - 6909 6915-6952; 6954 -6994 7016-7037 7040 - 7060 7082 - 7096 7108-7142 7158-7173 7175-7223; 7225-7242 7244 - 7298 7300-7317 7319 -7397 7399 - 7452 7454 - 7487 7490 -7518; 7525 -7550 7557 - 7576 7587 - 7601 7625-7641 7640 - 7666 7670 - 7684 7704 -7761; 7763 -7816 7818-7871 7893 - 7907 7919 -7938 7940 - 7984 7986 -8011 8013 -8044; 8040 -8062 8052 - 8067 8069 - 8098 8100-8148 8150 -8201 8215-8233 8249 - 8288; 8297 - 8327 8352 - 8368 8372 - 8386 8391 -8438 8442 - 8465 8469 - 8524 8526 - 8546; 8549 - 8573 8603 - 8634 8670 - 8684 8727 - 8751 8753 - 8767 8773 - 8788 8836 - 8852; 8854 - 8869 8885-8916 8918-8944 8975 - 8989 9002 - 9026 9028 - 9050 9065-9096; 9099 -9128 9130-9202 9219-9237 9226 - 9244 9241 - 9275 9288 -9331 9333 - 9399; 9389 - 9420 9438 - 9498 9500-9518 9529 - 9545 9554 - 9573 9570 - 9605 9645 - 9663; 9709 - 9733 9764 - 9785 9801 - 9834 9847 - 9864 9866 - 9880 9882 - 9900 9920 - 9968; 9970 - 9990 10008 - 10022; 10024 - 10053; 10066 - 10085; 10098- 10114; 10126-10151; 10155- 10169; 10183-10197 10202 - 10222; 10224 - 10291; 10293 - 10308; 10306 - 10320; 10322 - 10337; 10344 10369; 10371 - 10397; 10416- 10477; 10496- 10521; 10515- 10530; 10523- 10546 10558 - 10580; 10576 - 10625; 10627 - 10702; 10705 - 10732; 10731 - 10748; 10750 10765; 10768 - 10783; 10792 - 10875; 10877 - 10908; 10917 - 10961; 10963 - 10977; 10970 - 10992; 11026- 11041; 11055- 11102; 11104- 11125; 11127- 11153; 11159- 11194; 11196-11215; 11273- 11290; 11310- 11372; 11395- 11435; 11461 - 11475; 11509- 11525; 11530- 11545; 11547- 11573; 11589- 11606; 11609- 11642; 11644- 11672; 11695- 11710; 11719 - 11749; 11781 - 11821; 11876- 11891; 11897- 11919; 11921 - 11942; 11944- 11974; 11987- 12010; 12039- 12068; 12090- 12106; 12132 - 12157; 12180 - 12194; 12206 - 12225; 12228 - 12246; 12242 - 12267; 12284 - 12302; 12298 - 12315; 12345 - 12362; 12364 - 12399; 12413 - 12432; 12451 - 12483; 12517 - 12549; 12556 - 12595; 12597 - 12619; 12621 - 12638; 12640 - 12672; 12687 - 12710; 12712 - 12727; 12734 - 12748; 12750 - 12788; 12807 - 12836; 12866 - 12880; 12891 - 12915; 12925 - 12962; 12968 - 12992; 12994 - 13013; 13031 - 13064; 13078 - 13094; 13103 - 13123; 13140 - 13155; 13154 - 13185; 13218 - 13259; 13273 - 13293; 13297 - 13319; 13361 - 13432; 13434 - 13452; 13465 - 13484; 13483 - 13533; 13546 - 13560; 13572 - 13596; 13607 - 13668; 13670 - 13690; 13691 - 13768; 13766 - 13783; 13821 - 13835; 13837 - 13863; 13882- 13911; 13932- 13952; 13953- 13973; 14029- 14067; 14069 - 14089; 14091 - 14134; 14156 - 14186; 14188 - 14203; 14248 - 14267; 14308 - 14324; 14326 - 14354; 14375 - 14392; 14398 - 14454; 14456 - 14479; 14481 - 14510; 14512 - 14543; 14545 - 14586; 14597 - 14646; 14648 - 14669; 14694 - 14724; 14763 - 14786; 14798 - 14816; 14845- 14871; 14887- 14904; 14938- 14953; 14955- 14980; 14991 - 15008; 15010 - 15032; 15053 - 15078; 15081 - 15095; 15126 - 15151; 15172 - 15200; 15225 - 15255; 15257 - 15288; 15290 - 15310; 15312 - 15356; 15359 - 15401; 15403 - 15417; 15430 - 15462; 15465 - 15500; 15502 - 15525; 15540 - 15558; 15568 - 15606; 15620 - 15659; 15661 - 15701; 15703 - 15728; 15730 - 15771; 15781 - 15807; 15809 - 15866; 15881 - 15904; 15906 - 15947; 15959 - 15975; 15993 - 16029; 16031 - 16082; 16084 - 16098; 16100 - 16122; 16124 - 16143; 16157 - 16172; 16174 - 16206; 16210 - 16234; 16243- 16269; 16283- 16309; 16322- 16336; 16353- 16381; 16390 - 16406; 16437 - 16470; 16515 - 16529; 16537 - 16567; 16579 - 16624; 16632 - 16679; 16758 - 16801; 16812 - 16847; 16859 - 16892; 16894 - 16908; 16910 - 16958; 16973 - 16994; 17038 - 17072; 17079 - 17099; 17127 - 17156; 17160 - 17184; 17186 - 17221; 17238 - 17254; 17256 - 17303; 17310 - 17341; 17354 - 17385; 17396 - 17419; 17432 - 17448; 17460 - 17480; 17482 - 17496; 17518 - 17536; 17538 - 17555; 17557 - 17588; 17593 - 17610; 17612- 17627; 17637- 17663; 17665- 17683; 17710- 17741; 17743- 17760; 17787 - 17834; 17839 - 17881 ; 17887 - 17923; 17925 - 17943; 17962 - 17998; 18000 - 18014; 18018- 18032; 18034- 18070; 18072- 18091; 18098- 18115; 18117- 18142; 18139 - 18179; 18182 - 18207; 18204 - 18224; 18256 - 18299; 18301 - 18345; 18347 - 18364; 18366 - 18397; 18396 - 18418; 18430 - 18459; 18468 - 18482; 18484 - 18513; 18515 - 18536; 18538 - 18567; 18603 - 18617; 18633 - 18652; 18688 - 18755; 18755 - 18778; 18794 - 18836; 18832 - 18864; 18880 - 18906; 18914 - 18938; 18933 - 18948; 18940 - 18963; 18973 - 18987; 18989 - 19007; 19039 - 19062; 19071 - 19097; 19099 - 19130; 19152 - 19184; 19189 - 19207; 19221 - 19255; 19257 - 19299; 19313 - 19327; 19325 - 19350; 19366 - 19391 ; 19423 - 19470; 19473 - 19528; 19574 - 19624; 19626 - 19647; 19649 - 19673; 19675 - 19707; 19720 - 19770; 19797 - 19812; 19814 - 19831 ; 19833 - 19892; 19924 - 19980; 19982 - 20001 ; 20003 - 20044; 20069 - 20084; 20095 - 20140; 20142 - 20186; 20204 - 20228; 20230 - 20262; 20270 - 20296; 20308 - 20322; 20332 - 20395; 20404 - 20471 ; 20503 - 20532; 20543 - 20578; 20580 - 20601 ; 20603 - 20625; 20653 - 20672; 20674 - 20703; 20705 - 20763; 20772 - 20792; 20798 - 20861 ; 20863 - 20893; 20884 - 20942; 20944 - 20964; 20984 - 21002; 21004 - 21056; 21058 - 21072; 21 106 - 21 133; 21 142 - 21 159; 21 161 - 21 177; 21 187 - 21217; 21218 - 21237; 21239 - 21281 ; 21290 - 21307; 21308 - 21341 ; 21343 - 21364; 21378 - 21402; 21407 - 21435; 21437 - 21462; 21501 - 21530; 21547 - 21565; 21567 - 21594; 21596 - 21613; 21615 - 21635; 21649 - 21694; 21699 - 21722; 21724 - 21774; 21785 - 21833; 22324 - 22339; 22334 - 22348; 22362 - 22381 ; 22404 - 22426; 22427 - 22463; 22463 - 22477; 22479 - 22515; 22515 - 22542; 22544 - 22570; 22572 - 22599; 22604 - 22620; 22634 - 22665; 22667 - 22688; 22702 - 22723; 22726 - 22740; 22750 - 22805; 22807 - 22828; 22851 - 22868; 22880 - 22913; 22960 - 22990; 23028 - 23092; 23107 - 23122; 23143 - 23182; 23192 - 23210; 23243 - 23268; 23276 - 23309; 23319 - 23402; 23408 - 23432; 23434 - 23458; 23460 - 23483; 23485 - 23506; 23529 - 23548; 23600 - 23621 ; 23664 - 23678; 23680 - 23699; 23701 - 23757; 23759 - 23804; 23831 - 23857; 23859 - 23878; 23916 - 23974; 23972 - 24009; 2401 1 - 24042; 24044 - 24063; 24078 - 24104; 24135 - 24159; 24192 - 24206; 24208 - 24230; 24232 - 24249; 24251 - 24282; 24284 - 24321 ; 24323 - 24346; 24357 - 24376; 24391 - 24455; 24457 - 24494; 24496 - 24522; 24524 - 24553; 24555 - 24576; 24578 - 24599; 24601 - 24620; 24622 - 24674; 24691 - 24711 ; 24713 - 24767; 24771 - 24786; 24793 - 24857; 24901 - 24993; 25005 - 25021 ; 25031 - 25049; 25055 - 25077; 25079 - 25094; 25096 - 25128; 25185 - 25207; 25209 - 25227; 25229 - 25261 ; 25263 - 25277; 25290 - 25339; 25368 - 25382; 25400 - 25431 ; 25433 - 25460; 25479 - 25523; 25538 - 25558; 25560 - 25588; 25607 - 25639; 25641 - 25665; 25671 - 25696; 25698 - 25725; 25727 - 25741 ; 25757 - 25788; 25790 - 25805; 25807 - 25826; 25828 - 25851 ; 25851 - 25871 ; 25910 - 25965; 25967 - 26022; 26039 - 26071 ; 26073 - 26090; 26105 - 26121 ; 26137 - 26155; 26155 - 26259; 26273 - 26324; 26336 - 26351 ; 26372 - 26386; 26413 - 26431 ; 26445 - 26460; 26477 - 26493; 2651 1 - 26538; 26541 - 26564; 26566 - 26592; 26609 - 26626; 26628 - 26656; 26690 - 26719; 26730 - 26746; 26748 - 26769; 26771 - 26791 ; 26793 - 26811 ; 26813 - 26852; 26869 - 26894; 26906 - 26924; 26930 - 26973; 26987 - 27010; 27012 - 27028; 27044 - 27075; 27078 - 27095; 27097 - 27119; 27146 - 27160; 27179 - 27199; 27201 - 27217; 27219 - 27233; 27243 - 27259; 27261 - 27284; 27290 - 27316; 27326 - 27345; 27365 - 27408; 27410 - 27437; 27486 - 27520; 27537 - 27562; 27620 - 27646; 27649 - 27670; 27675 - 27712; 27714 - 27736; 27753 - 27796; 27798 - 27832; 27852 - 27869; 27871 - 27902; 27911 -27930; 27970-28015; 28018-28124; 28128-28174; 28176-28193; 28195- 28221 ; 28232 - 28283; 28301 - 28323; 28331 - 28358; 28364 - 28388; 28421 - 28452; 28454 - 28500; 28528 - 28548; 28560 - 28717; 28719 - 28760; 28762 - 28778; 28795 - 28829; 28831 - 28852; 28898 - 28959; 28964 - 28994; 28996 - 29022; 29041 - 29067; 29088 -29117; 29119 - 29151; 29153 - 29197; 29199-29226; 29252-29290; 29405- 29435; 29439 - 29456; 29463 - 29501 ; 29503 - 29524; 29537 - 29551 ; 29557 - 29575; 29582 - 29597; 29611 - 29636; 29638 - 29666; 29668 - 29683; 29706 - 29730; 29732 - 29757; 29760 - 29856; 29858 - 29895; 29897 - 29958; 29974 - 30003; 30005 - 30050; 30052 -30089; 30091 -30108; 30110-30134; 30136-30160; 30176-30190; 30192- 30227; 30233 - 30255; 30261 - 30308; 30310 - 30330; 30347 - 30368; 30420 - 30441; 30443 - 30468; 30470 - 30494; 30519 - 30533; 30535 - 30558; 30560 - 30588; 30592 - 30622; 30635 - 30653; 30652 - 30672; 30674 - 30703; 30712 - 30733; 30738 - 30786; 30807 - 30822; 30854 - 30879; 30881 - 30942; 30944 - 30964; 30964 - 31003; 31004 - 31026; 31028 -31057; 31066-31085; 31087-31116; 31118-31182; 31184-31201; 31203 - 31224; 31239 - 31262; 31268 - 31307; 31312 - 31330; 31336 - 31352; 31360 - 31392; 31417 - 31434; 31439 - 31466; 31468 - 31501; 31530 - 31544; 31536 - 31575; 31577 -31594; 31596-31634; 31633-31650; 31651 -31675; 31680-31714; 31711 - 31725; 31743 - 31758; 31762 - 31805; 31837 - 31853; 31855 - 31874; 31881 - 31899; 31935 - 31951; 31953 - 31977; 31979 - 32073; 32106 - 32123; 32126 - 32146; 32143 - 32162; 32168 - 32199; 32199 - 32227; 32229 - 32262; 32262 - 32278; 32280 - 32315; 32317 - 32363; 32390 - 32441; 32452 - 32479; 32481 - 32496; 32534 - 32603; 32605 - 32632; 32647 - 32694; 32711 - 32750; 32797 - 32826; 32828 - 32861; 32870 - 32885; 32906 - 32926; 32935 - 32952; 32979 - 33002; 33004 - 33037; 33057 - 33084; 33086 - 33109; 33111 -33139; 33150-33191; 33207-33233; 33235-33251; 33288-33355; 33355 - 33411 ; 33425 - 33452; 33460 - 33489; 33492 - 33536; 33536 - 33557; 33562 - 33584; 33586 - 33604; 33606 - 33623; 33631 - 33659; 33661 - 33679; 33681 - 33707; 33730 - 33765; 33814 - 33828; 33844 - 33861; 33875 - 33915; 33936 - 33958; 33999 - 34027; 34049 - 34244; 34270 - 34292; 34299 - 34359; 34371 - 34586; 34592 - 34642; 34644 - 34667; 34669 - 34737; 34739 - 34756; 34789 - 34813; 34815 - 34835; 34839 - 34853; 34868 - 34904; 34912 - 34927; 34946 - 34963; 34976 - 35013; 35015 - 35030; 35083 - 35142; 35157 - 35192; 3521 1 - 35234; 35253 - 35307; 35340 - 35370; 35392 - 35411 ; 35447 - 35470; 35491 - 35515; 35526 - 35593; 35651 - 35682; 35742 - 35760; 35776 - 35792; 35812 - 35899; 35901 - 35943; 35945 - 35979; 35988 - 36019; 36033 - 36047; 36067 - 36101 ; 36117 - 36141 ; 36143 - 36159; 36206 - 36228; 36230 - 36253; 36255 - 36284; 36286 - 36336; 36339 - 36353; 36355 - 36393; 36408 - 36481 ; 36501 - 36524; 36526 - 36549; 36551 - 36582; 36585 - 36607; 36616 - 36702; 36704 - 36752; 36750 - 36782; 36790 - 36835; 36837 - 36873; 36878 - 36902; 36904 - 36924; 36926 - 36962; 36973 - 36988; 36990 - 37012; 37049 - 37074; 37076 - 37115; 37152 - 37192; 37210 - 37225; 37227 - 37260; 37275 - 37307; 37324 - 37349; 37361 - 37381 ; 37388 - 37407; 37415 - 37454; 37474 - 37519; 37530 - 37572; 37572 - 37605; 37617 - 37656; 37658 - 37685; 37720 - 37787; 37817 - 37879; 37885 - 37923; 37925 - 37943; 37966 - 37985; 38002 - 38032; 38039 - 38064; 38066 - 38135; 38148 - 38180; 38214 - 38276; 38278 - 38310; 38314 - 38337; 38346 - 38404; 38406 - 38459; 38461 - 38487; 38509 - 38551 ; 38553 - 38590; 38592 - 38614; 38616 - 38633; 38635 - 38692; 38712 - 38747; 38749 - 38773; 38775 - 38797; 3881 1 - 38846; 38849 - 38890; 38900 - 38917; 38942 - 38964; 38966 - 38998; 39000 - 39069; 39098 - 39115; 39117 - 39157; 39252 - 39297; 39300 - 39358; 39393 - 39415; 39435 - 39488; 39494 - 3951 1 ; 39529 - 39543; 39592 - 39607; 39625 - 39657; 39685 - 39702; 39713 - 39730; 39732 - 39765; 39767 - 39788; 39797 - 39835; 39837 - 39866; 39868 - 39900; 39909 - 39929; 39929 - 39956; 39969 - 40004; 40006 - 40025; 40027 - 40069; 40071 - 40095; 40134 - 40157; 40175 - 40198; 40216 - 40255; 40257 - 40306; 40319 - 40339; 40354 - 40376; 40379 - 40413; 40443 - 40485; 40496 - 40514; 40531 - 40550; 40552 - 40569; 40571 - 40588; 40602 - 40635; 40641 - 40666; 40706 - 40835; 40837 - 40854; 40856 - 40878; 40880 - 40896; 40917 - 40959; 40961 - 40988; 40990 - 41025; 41058 - 41074; 41087 - 41 110; 41 123 - 41 149; 41151 - 41225; 41255 - 41300; 41310 - 41331 ; 41333 - 41347; 41349 - 41363; 41365 - 41387; 41389 - 41405; 41417 - 41437; 41439 - 41453; 41461 - 41488; 41542 - 41561 ; 41563 - 41587; 41594 - 41620; 41622 - 41654; 41666 - 41682; 41689 - 41713; 41729 - 41808; 41810 - 41828; 41833 - 41854; 41861 - 41883; 41888 - 41925; 41927 - 41950; 41963 - 41979; 41981 - 42004; 42009 - 42030; 42048 - 42070; 42072 - 42088; 42096 - 42123; 42181 - 42195; 42228 - 42247; 42304 - 42328; 42337 - 42356; 42358 - 42390; 42404 - 42419; 42428 - 42442; 42448 - 42497; 42505 - 42565; 42597 - 42646; 42660 - 42686; 42686 - 42701 ; 42703 - 42741 ; 42760 - 42816; 42818 - 42880; 42882 - 42940; 42983 - 43028; 43060 - 43077; 43093 - 43108; 43110 - 43156; 43171 - 43215; 43217 - 43233; 43255 - 43276; 43282 - 43304; 43336 - 43351 ; 43356 - 43387; 43389 - 43434;

43436 - 43498; 43500 - 43694; 43696 - 43730; 43732 - 43749; 43752 - 43786; 43837 - 43865; 43875 - 43927; 43966 - 43980; 43996 - 44013; 44025 - 44081; 44089-44111;

44156 - 44182; 44201 - 44218; 44220 - 44255; 44269 - 44290; 44316 -44341; 44355- 44389; 44391 - 44417; 44419 - 44448; 44450 - 44473; 44481 - 44507; 44508 - 44528;

44530 - 44545; 44547 - 44597; 44636 - 44653; 44665 - 44697; 44711 - 44795; 44797 - 44817; 44819 - 44876; 44882 - 44901; 44914-44943; 44945- 44963; 44985-45010;

45013 - 45057; 45059 - 45097; 45099-45119; 45121 -45166; 45176-45212; 45229- 45254; 45256 - 45280; 45283 - 45364; 45366 - 45402; 45423 - 45480; 45499 - 45572;

45574 - 45597; 45604 - 45622; 45635 - 45662; 45664 - 45687; 45689 - 45711 ; 45713- 45760; 45769 - 45809; 45820 - 45873; 45875 - 45959; 45987 - 46031; 46052-46077;

46082 -46113; 46116-46133; 46146-46176; 46191 -46226; 46247 - 46263; 46265 - 46280; 46298 - 46325; 46327 - 46344; 46353 - 46383; 46395 - 46410; 46441 -46457;

46482 -46511; 46516 -46533; 46548 - 46573; 46619 - 46675; 46688-46705; 46717- 46732; 46739 - 46756; 46758 - 46777; 46779 - 46797; 46809 - 46831; 46833-46875;

46877 - 46917; 46941 - 46958; 46975 - 46990; 47007 - 47030; 47032 - 47063; 47065 - 47103; 47105 -47135; 47143 - 47190; 47195 - 47236; 47238 - 47263; 47269-47311;

47313 - 47333; 47335 - 47356; 47388 - 47403; 47405 - 47441 ; 47437 - 47456; 47469 - 47509; 47521 - 47572; 47570 - 47595; 47597 - 47619; 47632 - 47780; 47820 - 47850;

47852 - 47874; 47880 - 48034; 48044-48108; 48111 -48133; 48143-48159; 48175- 48191 ; 48193 - 48227; 48245 - 48268; 48282 - 48312; 48316 - 48339; 48369 - 48387;

48383 - 48418; 48441 - 48466; 48474 - 48533; 48540 - 48599; 48687 - 48726; 48728 - 48760; 48792 - 48827; 48829 - 48860; 48863 - 48903; 48905 - 49023; and 49025 - 49058.

Target Cell

The term a“target cell” as used herein refers to a cell which is expressing the target nucleic acid. In some embodiments the target cell may be in vivo or in vitro. In some embodiments the target cell is a mammalian cell such as a rodent cell, such as a mouse cell or a rat cell, or a primate cell such as a monkey cell or a human cell.

In preferred embodiments the target cell expresses BCL2L11 mRNA, such as the BCL2L11 pre-mRNA, e.g. SEQ ID NO 15, or BCL2L11 mature mRNA (e.g. SEQ ID NO 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32 or 33). The poly A tail of BCL2L11 mRNA is typically disregarded for antisense oligonucleotide targeting. Naturally occurring variant

The term“naturally occurring variant” refers to variants of BCL2L11 gene or transcripts which originate from the same genetic loci as the target nucleic acid, but may differ for example, by virtue of degeneracy of the genetic code causing a multiplicity of codons encoding the same amino acid, or due to alternative splicing of pre-mRNA, or the presence of polymorphisms, such as single nucleotide polymorphisms (SNPs), and allelic variants. Based on the presence of the sufficient complementary sequence to the oligonucleotide, the oligonucleotide of the invention may therefore target the target nucleic acid and naturally occurring variants thereof.

The homo sapiens BCL2L1 1 gene is located at chromosome 2, 1 1 1 120914...1 1 1 168445, complement (NC_000002.12, Gene ID 10018).

In some embodiments, the naturally occurring variants have at least 95% such as at least 98% or at least 99% homology to a mammalian BCL2L11 target nucleic acid, such as a target nucleic acid selected form the group consisting of SEQ ID NO 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32 or 33. In some embodiments the naturally occurring variants have at least 99% homology to the human BCL2L11 target nucleic acid of SEQ ID NO 15.

Modulation of expression

The term“modulation of expression” as used herein is to be understood as an overall term for an oligonucleotide’s ability to alter the amount of BCL2L1 1 protein or BCL2L11 mRNA when compared to the amount of BCL2L1 1 or BCL2L11 mRNA prior to administration of the oligonucleotide. Alternatively modulation of expression may be determined by reference to a control experiment. It is generally understood that the control is an individual or target cell treated with a saline composition or an individual or target cell treated with a non-targeting oligonucleotide (mock).

One type of modulation is an oligonucleotide’s ability to inhibit, down-regulate, reduce, suppress, remove, stop, block, prevent, lessen, lower, avoid or terminate expression of BCL2L1 1 , e.g. by degradation of BCL2L11 mRNA.

High affinity modified nucleosides

A high affinity modified nucleoside is a modified nucleotide which, when incorporated into the oligonucleotide enhances the affinity of the oligonucleotide for its complementary target, for example as measured by the melting temperature (T^m). A high affinity modified nucleoside of the present invention preferably result in an increase in melting temperature between +0.5 to +12°C, more preferably between +1.5 to +10°C and most preferably between+3 to +8°C per modified nucleoside. Numerous high affinity modified nucleosides are known in the art and include for example, many 2’ substituted nucleosides as well as locked nucleic acids (LNA) (see e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213).

Sugar modifications

The oligomer of the invention may comprise one or more nucleosides which have a modified sugar moiety, i.e. a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA.

Numerous nucleosides with modification of the ribose sugar moiety have been made, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and/or nuclease resistance.

Such modifications include those where the ribose ring structure is modified, e.g. by replacement with a hexose ring (HNA), or a bicyclic ring, which typically have a biradicle bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g. UNA). Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids (WO201 1/017521 ) or tricyclic nucleic acids (WO2013/154798). Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.

Sugar modifications also include modifications made via altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2’-OH group naturally found in DNA and RNA nucleosides. Substituents may, for example be introduced at the 2’, 3’, 4’ or 5’ positions.

2’ sugar modified nucleosides.

A 2’ sugar modified nucleoside is a nucleoside which has a substituent other than H or -OH at the 2’ position (2’ substituted nucleoside) or comprises a 2’ linked biradicle capable of forming a bridge between the 2’ carbon and a second carbon in the ribose ring, such as LNA (2’ - 4’ biradicle bridged) nucleosides.

Indeed, much focus has been spent on developing 2’ substituted nucleosides, and numerous 2’ substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides. For example, the 2’ modified sugar may provide enhanced binding affinity and/or increased nuclease resistance to the oligonucleotide. Examples of 2’ substituted modified nucleosides are 2’-0-alkyl-RNA, 2’-0-methyl-RNA, 2’- alkoxy-RNA, 2’-0-methoxyethyl-RNA (MOE), 2’-amino-DNA, 2’-Fluoro-RNA, and 2’-F-ANA nucleoside. For further examples, please see e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are illustrations of some 2’ substituted modified nucleosides.

In relation to the present invention 2’ substituted does not include 2’ bridged molecules like LNA.

Locked Nucleic Acids (LNA)

A“LNA nucleoside” is a 2’- modified nucleoside which comprises a biradical linking the C2’ and C4’ of the ribose sugar ring of said nucleoside (also referred to as a“2’- 4’ bridge”), which restricts or locks the conformation of the ribose ring. These nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature. The locking of the conformation of the ribose is associated with an enhanced affinity of hybridization (duplex stabilization) when the LNA is incorporated into an oligonucleotide for a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide/complement duplex.

Non limiting, exemplary LNA nucleosides are disclosed in WO 99/014226, WO

00/66604, WO 98/039352 , WO 2004/046160, WO 00/047599, WO 2007/134181 , WO 2010/077578, WO 2010/036698, WO 2007/090071 , WO 2009/006478, WO 2011/156202, WO 2008/154401 , WO 2009/067647, WO 2008/150729, Morita et al., Bioorganic &

Med.Chem. Lett. 12, 73-76, Seth et al. J. Org. Chem. 2010, Vol 75(5) pp. 1569-81 , and Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667.

Further non limiting, exemplary LNA nucleosides are disclosed in Scheme 1. Scheme 1 :

Particular LNA nucleosides are beta-D-oxy-LNA, 6’-methyl-beta-D-oxy LNA such as (S)-6’-methyl-beta-D-oxy-LNA (ScET) and ENA.

A particularly advantageous LNA is beta-D-oxy-LNA.

RNase H Activity and Recruitment

The RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule. WO01/23613 provides in vitro methods for determining RNaseH activity, which may be used to determine the ability to recruit RNaseH. Typically an oligonucleotide is deemed capable of recruiting RNase H if it, when provided with a complementary target nucleic acid sequence, has an initial rate, as measured in pmol/l/min, of at least 5%, such as at least 10% or more than 20% of the of the initial rate determined when using a oligonucleotide having the same base sequence as the modified oligonucleotide being tested, but containing only DNA monomers with

phosphorothioate linkages between all monomers in the oligonucleotide, and using the methodology provided by Example 91 - 95 of WO01/23613 (hereby incorporated by reference). For use in determining RHase H activity, recombinant human RNase H1 is available from Lubio Science GmbH, Lucerne, Switzerland. Gapmer

The antisense oligonucleotide of the invention, or contiguous nucleotide sequence thereof may be a gapmer. The antisense gapmers are commonly used to inhibit a target nucleic acid via RNase H mediated degradation. A gapmer oligonucleotide comprises at least three distinct structural regions a 5’-flank, a gap and a 3’-flank, F-G-F’ in the‘5 -> 3’ orientation. The“gap” region (G) comprises a stretch of contiguous DNA nucleotides which enable the oligonucleotide to recruit RNase H. The gap region is flanked by a 5’ flanking region (F) comprising one or more sugar modified nucleosides, advantageously high affinity sugar modified nucleosides, and by a 3’ flanking region (F’) comprising one or more sugar modified nucleosides, advantageously high affinity sugar modified nucleosides. The one or more sugar modified nucleosides in region F and F’ enhance the affinity of the oligonucleotide for the target nucleic acid ( i.e . are affinity enhancing sugar modified nucleosides). In some embodiments, the one or more sugar modified nucleosides in region F and F’ are 2’ sugar modified nucleosides, such as high affinity 2’ sugar modifications, such as independently selected from LNA and 2’-MOE.

In a gapmer design, the 5’ and 3’ most nucleosides of the gap region are DNA nucleosides, and are positioned adjacent to a sugar modified nucleoside of the 5’ (F) or 3’ (F’) region respectively. The flanks may further defined by having at least one sugar modified nucleoside at the end most distant from the gap region, i.e. at the 5’ end of the 5’ flank and at the 3’ end of the 3’ flank.

Regions F-G-F’ form a contiguous nucleotide sequence. Antisense oligonucleotides of the invention, or the contiguous nucleotide sequence thereof, may comprise a gapmer region of formula F-G-F’.

The overall length of the gapmer design F-G-F’ may be, for example 12 to 32 nucleosides, such as 13 to 24, such as 14 to 22 nucleosides, Such as from 14 to17, such as 16 to18 nucleosides.

By way of example, the gapmer oligonucleotide of the present invention can be represented by the following formulae:

FI-8-G₅-I6-F’I-₈, such as

F1-8-G7-16-F 2-8

with the proviso that the overall length of the gapmer regions F-G-F’ is at least 12, such as at least 14 nucleotides in length.

Regions F, G and F’ are further defined below and can be incorporated into the F-G-F’ formula. Gapmer - Region G

Region G (gap region) of the gapmer is a region of nucleosides which enables the oligonucleotide to recruit RNaseH, such as human RNase H1 , typically DNA nucleosides. RNaseH is a cellular enzyme which recognizes the duplex between DNA and RNA, and enzymatically cleaves the RNA molecule. Suitably gapmers may have a gap region (G) of at least 5 or 6 contiguous DNA nucleosides, such as 5 - 16 contiguous DNA nucleosides, such as 6 - 15 contiguous DNA nucleosides, such as 7-14 contiguous DNA nucleosides, such as 8 - 12 contiguous DNA nucleotides, such as 8 - 12 contiguous DNA nucleotides in length. The gap region G may, in some embodiments consist of 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or 16 contiguous DNA nucleosides. One or more cytosine (C) DNA in the gap region may in some instances be methylated (e.g. when a DNA c is followed by a DNA g) such residues are either annotated as 5-methyl-cytosine (^meC). In some embodiments the gap region G may consist of 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 contiguous phosphorothioate linked DNA nucleosides. In some embodiments, all internucleoside linkages in the gap are phosphorothioate linkages.

Whilst traditional gapmers have a DNA gap region, there are numerous examples of modified nucleosides which allow for RNaseH recruitment when they are used within the gap region. Modified nucleosides which have been reported as being capable of recruiting RNaseH when included within a gap region include, for example, alpha-L-LNA, C4’ alkylated DNA (as described in PCT/EP2009/050349 and Vester et a!., Bioorg. Med. Chem. Lett. 18 (2008) 2296 - 2300, both incorporated herein by reference), arabinose derived nucleosides like ANA and 2'F-ANA (Mangos et al. 2003 J. AM. CHEM. SOC. 125, 654-661 ), UNA

(unlocked nucleic acid) (as described in Fluiter et al., Mol. Biosyst, 2009, 10, 1039 incorporated herein by reference). UNA is unlocked nucleic acid, typically where the bond between C2 and C3 of the ribose has been removed, forming an unlocked“sugar” residue. The modified nucleosides used in such gapmers may be nucleosides which adopt a 2’ endo (DNA like) structure when introduced into the gap region, i.e. modifications which allow for RNaseH recruitment). In some embodiments the DNA Gap region (G) described herein may optionally contain 1 to 3 sugar modified nucleosides which adopt a 2’ endo (DNA like) structure when introduced into the gap region.

Region G -“Gap-breaker”

Alternatively, there are numerous reports of the insertion of a modified nucleoside which confers a 3’ endo conformation into the gap region of gapmers, whilst retaining some RNaseH activity. Such gapmers with a gap region comprising one or more 3’endo modified nucleosides are referred to as“gap-breaker” or“gap-disrupted” gapmers, see for example WO2013/022984. Gap-breaker oligonucleotides retain sufficient region of DNA nucleosides within the gap region to allow for RNaseH recruitment. The ability of gapbreaker

oligonucleotide design to recruit RNaseH is typically sequence or even compound specific - see Rukov et al. 2015 Nucl. Acids Res. Vol. 43 pp. 8476-8487, which discloses“gapbreaker” oligonucleotides which recruit RNaseH which in some instances provide a more specific cleavage of the target RNA. Modified nucleosides used within the gap region of gap- breaker oligonucleotides may for example be modified nucleosides which confer a 3’endo confirmation, such 2’ -O-methyl (OMe) or 2’-0-MOE (MOE) nucleosides, or beta-D LNA nucleosides (the bridge between C2’ and C4’ of the ribose sugar ring of a nucleoside is in the beta conformation), such as beta-D-oxy LNA or ScET nucleosides.

As with gapmers containing region G described above, the gap region of gap-breaker or gap-disrupted gapmers, have a DNA nucleosides at the 5’ end of the gap (adjacent to the 3’ nucleoside of region F), and a DNA nucleoside at the 3’ end of the gap (adjacent to the 5’ nucleoside of region F’). Gapmers which comprise a disrupted gap typically retain a region of at least 3 or 4 contiguous DNA nucleosides at either the 5’ end or 3’ end of the gap region. Exemplary designs for gap-breaker oligonucleotides include

Fl-8-[D3-4-El- D 3-₄]-F ¹ ₁ -8

wherein region G is within the brackets [D_n-E_r- D_m], D is a contiguous sequence of DNA nucleosides, E is a modified nucleoside (the gap-breaker or gap-disrupting nucleoside), and F and F’ are the flanking regions as defined herein, and with the proviso that the overall length of the gapmer regions F-G-F’ is at least 12, such as at least 14 nucleotides in length. In some embodiments, region G of a gap disrupted gapmer comprises at least 6 DNA nucleosides, such as 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 DNA nucleosides. As described above, the DNA nucleosides may be contiguous or may optionally be interspersed with one or more modified nucleosides, with the proviso that the gap region G is capable of mediating RNaseH recruitment.

Gapmer - flanking regions, F and F’

Region F is positioned immediately adjacent to the 5’ DNA nucleoside of region G. The 3’ most nucleoside of region F is a sugar modified nucleoside, such as a high affinity sugar modified nucleoside, for example a 2’ substituted nucleoside, such as a MOE nucleoside, or an LNA nucleoside.

Region F’ is positioned immediately adjacent to the 3’ DNA nucleoside of region G. The 5’ most nucleoside of region F’ is a sugar modified nucleoside, such as a high affinity sugar modified nucleoside, for example a 2’ substituted nucleoside, such as a MOE nucleoside, or an LNA nucleoside.

Region F is 1 - 8 contiguous nucleotides in length, such as 2-6, such as 3-4 contiguous nucleotides in length. Advantageously the 5’ most nucleoside of region F is a sugar modified nucleoside. In some embodiments the two 5’ most nucleoside of region F are sugar modified nucleoside. In some embodiments the 5’ most nucleoside of region F is an LNA nucleoside. In some embodiments the two 5’ most nucleoside of region F are LNA nucleosides. In some embodiments the two 5’ most nucleoside of region F are 2’ substituted nucleoside nucleosides, such as two 3’ MOE nucleosides. In some embodiments the 5’ most nucleoside of region F is a 2’ substituted nucleoside, such as a MOE nucleoside.

Region F’ is 2 - 8 contiguous nucleotides in length, such as 3-6, such as 4-5 contiguous nucleotides in length. Advantageously, embodiments the 3’ most nucleoside of region F’ is a sugar modified nucleoside. In some embodiments the two 3’ most nucleoside of region F’ are sugar modified nucleoside. In some embodiments the two 3’ most nucleoside of region F’ are LNA nucleosides. In some embodiments the 3’ most nucleoside of region F’ is an LNA nucleoside. In some embodiments the two 3’ most nucleoside of region F’ are 2’ substituted nucleoside nucleosides, such as two 3’ MOE nucleosides. In some embodiments the 3’ most nucleoside of region F’ is a 2’ substituted nucleoside, such as a MOE nucleoside. It should be noted that when the length of region F or F’ is one, it is advantageously an LNA nucleoside.

In some embodiments, region F and F’ independently consists of or comprises a contiguous sequence of sugar modified nucleosides. In some embodiments, the sugar modified nucleosides of region F may be independently selected from 2’-0-alkyl-RNA units, 2’-0- methyl-RNA, 2’-amino-DNA units, 2’-fluoro-DNA units, 2’-alkoxy-RNA, MOE units, LNA units, arabino nucleic acid (ANA) units and 2’-fluoro-ANA units.

In some embodiments, region F and F’ independently comprises both LNA and a 2’ substituted modified nucleosides (mixed wing design).

In some embodiments, region F and F’ consists of only one type of sugar modified nucleosides, such as only MOE or only beta-D-oxy LNA or only ScET. Such designs are also termed uniform flanks or uniform gapmer design.

In some embodiments, all the nucleosides of region F or F’, or F and F’ are LNA

nucleosides, such as independently selected from beta-D-oxy LNA, ENA or ScET

nucleosides. In some embodiments region F consists of 1-5, such as 2-4, such as 3-4 such as 1 , 2, 3, 4 or 5 contiguous LNA nucleosides. In some embodiments, all the nucleosides of region F and F’ are beta-D-oxy LNA nucleosides. In some embodiments, all the nucleosides of region F or F’, or F and F’ are 2’ substituted nucleosides, such as OMe or MOE nucleosides. In some embodiments region F consists of 1 , 2, 3, 4, 5, 6, 7, or 8 contiguous OMe or MOE nucleosides. In some embodiments only one of the flanking regions can consist of 2’ substituted nucleosides, such as OMe or MOE nucleosides. In some embodiments it is the 5’ (F) flanking region that consists 2’ substituted nucleosides, such as OMe or MOE nucleosides whereas the 3’ (F’) flanking region comprises at least one LNA nucleoside, such as beta-D-oxy LNA nucleosides or cET nucleosides. In some embodiments it is the 3’ (F’) flanking region that consists 2’ substituted nucleosides, such as OMe or MOE nucleosides whereas the 5’ (F) flanking region comprises at least one LNA nucleoside, such as beta-D-oxy LNA nucleosides or cET nucleosides.

In some embodiments, all the modified nucleosides of region F and F’ are LNA nucleosides, such as independently selected from beta-D-oxy LNA, ENA or ScET nucleosides, wherein region F or F’, or F and F’ may optionally comprise DNA nucleosides (an alternating flank, see definition of these for more details). In some embodiments, all the modified nucleosides of region F and F’ are beta-D-oxy LNA nucleosides, wherein region F or F’, or F and F’ may optionally comprise DNA nucleosides (an alternating flank, see definition of these for more details).

In some embodiments the 5’ most and the 3’ most nucleosides of region F and F’ are LNA nucleosides, such as beta-D-oxy LNA nucleosides or ScET nucleosides.

In some embodiments, the internucleoside linkage between region F and region G is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage between region F’ and region G is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkages between the nucleosides of region F or F’, F and F’ are phosphorothioate internucleoside linkages.

LNA Gapmer

An LNA gapmer is a gapmer wherein either one or both of region F and F’ comprises or consists of LNA nucleosides. A beta-D-oxy gapmer is a gapmer wherein either one or both of region F and F’ comprises or consists of beta-D-oxy LNA nucleosides.

In some embodiments the LNA gapmer is of formula: [LNA]i_₅-[region G] -[LNA]_I-5, wherein region G is as defined in the Gapmer region G definition.

MOE Gapmers

A MOE gapmers is a gapmer wherein regions F and F’ consist of MOE nucleosides. In some embodiments the MOE gapmer is of design [MOE]i-e-[Region G]-[MOE] 1-8, such as [MOE]2-7-[Region G]s-i₆-[MOE] 2-7, such as [MOE]3-6-[Region G]-[MOE] 3-6, wherein region G is as defined in the Gapmer definition. MOE gapmers with a 5-10-5 design (MOE-DNA-MOE) have been widely used in the art.

Mixed Wing Gapmer

A mixed wing gapmer is an LNA gapmer wherein one or both of region F and F’ comprise a 2’ substituted nucleoside, such as a 2’ substituted nucleoside independently selected from the group consisting of 2’-0-alkyl-RNA units, 2’-0-methyl-RNA, 2’-amino-DNA units, 2’- fluoro-DNA units, 2’-alkoxy-RNA, MOE units, arabino nucleic acid (ANA) units and 2’-fluoro- ANA units, such as a MOE nucleosides. In some embodiments wherein at least one of region F and F’, or both region F and F’ comprise at least one LNA nucleoside, the remaining nucleosides of region F and F’ are independently selected from the group consisting of MOE and LNA. In some embodiments wherein at least one of region F and F’, or both region F and F’ comprise at least two LNA nucleosides, the remaining nucleosides of region F and F’ are independently selected from the group consisting of MOE and LNA. In some mixed wing embodiments, one or both of region F and F’ may further comprise one or more DNA nucleosides.

Mixed wing gapmer designs are disclosed in W02008/049085 and WO2012/109395, both of which are hereby incorporated by reference.

Alternating Flank Gapmers

Oligonucleotides with alternating flanks are LNA gapmer oligonucleotides where at least one of the flanks (F or F’) comprises DNA in addition to the LNA nucleoside(s). In some embodiments at least one of region F or F’, or both region F and F’, comprise both LNA nucleosides and DNA nucleosides. In such embodiments, the flanking region F or F’, or both F and F’ comprise at least three nucleosides, wherein the 5’ and 3’ most nucleosides of the F and/or F’ region are LNA nucleosides.

In some embodiments at least one of region F or F’, or both region F and F’, comprise both LNA nucleosides and DNA nucleosides. In such embodiments, the flanking region F or F’, or both F and F’ comprise at least three nucleosides, wherein the 5’ and 3’ most nucleosides of the F or F’ region are LNA nucleosides, and there is at least one DNA nucleoside positioned between the 5’ and 3’ most LNA nucleosides of region F or F’ (or both region F and F’).

Region D’ or D” in an oligonucleotide

The oligonucleotide of the invention may in some embodiments comprise or consist of the contiguous nucleotide sequence of the oligonucleotide which is complementary to the target nucleic acid, such as the gapmer F-G-F’, and further 5’ and/or 3’ nucleosides. The further 5’ and/or 3’ nucleosides may or may not be fully complementary to the target nucleic acid.

Such further 5’ and/or 3’ nucleosides may be referred to as region D’ and D” herein.

The addition of region D’ or D” may be used for the purpose of joining the contiguous nucleotide sequence, such as the gapmer, to a conjugate moiety or another functional group. When used for joining the contiguous nucleotide sequence with a conjugate moiety is can serve as a biocleavable linker. Alternatively it may be used to provide exonucleoase protection or for ease of synthesis or manufacture.

Region D’ and D” can be attached to the 5’ end of region F or the 3’ end of region F’, respectively to generate designs of the following formulas D’-F-G-F’, F-G-F’-D” or

D’-F-G-F’-D”. In this instance the F-G-F’ is the gapmer portion of the oligonucleotide and region D’ or D” constitute a separate part of the oligonucleotide.

Region D’ or D” may independently comprise or consist of 1 , 2, 3, 4 or 5 additional nucleotides, which may be complementary or non-complementary to the target nucleic acid. The nucleotide adjacent to the F or F’ region is not a sugar-modified nucleotide, such as a DNA or RNA or base modified versions of these. The D’ or D’ region may serve as a nuclease susceptible biocleavable linker (see definition of linkers). In some embodiments the additional 5’ and/or 3’ end nucleotides are linked with phosphodiester linkages, and are DNA or RNA. Nucleotide based biocleavable linkers suitable for use as region D’ or D” are disclosed in WO2014/076195, which include by way of example a phosphodiester linked DNA dinucleotide. The use of biocleavable linkers in poly-oligonucleotide constructs is disclosed in WO2015/113922, where they are used to link multiple antisense constructs (e.g. gapmer regions) within a single oligonucleotide.

In one embodiment the oligonucleotide of the invention comprises a region D’ and/or D” in addition to the contiguous nucleotide sequence which constitutes the gapmer.

In some embodiments, the oligonucleotide of the present invention can be represented by the following formulae:

F-G-F’; in particular F1-8-G5-16-F 2-8

D’-F-G-F’, in particular D’i_-3-Fi-8-G5-i6-F’2-8

F-G-F’-D”, in particular Fi-8-G5-i6-F’2-8-D”i_-3

D’-F-G-F’-D”, in particular D’_I-3- Fi-8-G5-i6-F’2-8-D”i_-3

In some embodiments the internucleoside linkage positioned between region D’ and region F is a phosphodiester linkage. In some embodiments the internucleoside linkage positioned between region F’ and region D” is a phosphodiester linkage. Conjugate

The term conjugate as used herein refers to an oligonucleotide which is covalently linked to a non-nucleotide moiety (conjugate moiety or region C or third region).

Conjugation of the oligonucleotide of the invention to one or more non-nucleotide moieties may improve the pharmacology of the oligonucleotide, e.g. by affecting the activity, cellular distribution, cellular uptake or stability of the oligonucleotide. In some embodiments the conjugate moiety modify or enhance the pharmacokinetic properties of the oligonucleotide by improving cellular distribution, bioavailability, metabolism, excretion, permeability, and/or cellular uptake of the oligonucleotide. In particular the conjugate may target the oligonucleotide to a specific organ, tissue or cell type and thereby enhance the effectiveness of the oligonucleotide in that organ, tissue or cell type. A the same time the conjugate may serve to reduce activity of the oligonucleotide in non-target cell types, tissues or organs, e.g. off target activity or activity in non-target cell types, tissues or organs.

In an embodiment, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g. bacterial toxins), vitamins, viral proteins (e.g. capsids) or combinations thereof.

Linkers

A linkage or linker is a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds. Conjugate moieties can be attached to the oligonucleotide directly or through a linking moiety (e.g. linker or tether). Linkers serve to covalently connect a third region, e.g. a conjugate moiety (Region C), to a first region, e.g. an oligonucleotide or contiguous nucleotide sequence or gapmer region F-G-F’ (region A).

In some embodiments of the invention the conjugate or oligonucleotide conjugate of the invention may optionally, comprise a linker region (second region or region B and/or region Y) which is positioned between the oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid (region A or first region) and the conjugate moiety (region C or third region).

Region B refers to biocleavable linkers comprising or consisting of a physiologically labile bond that is cleavable under conditions normally encountered or analogous to those encountered within a mammalian body. Conditions under which physiologically labile linkers undergo chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidative or reductive conditions or agents, and salt concentration found in or analogous to those encountered in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activity normally present in a mammalian cell such as from proteolytic enzymes or hydrolytic enzymes or nucleases. In one embodiment the biocleavable linker is susceptible to S1 nuclease cleavage. DNA phosphodiester containing biocleavable linkers are described in more detail in WO 2014/076195 (hereby incorporated by reference) - see also region D’ or D” herein.

Region Y refers to linkers that are not necessarily biocleavable but primarily serve to covalently connect a conjugate moiety (region C or third region), to an oligonucleotide (region A or first region). The region Y linkers may comprise a chain structure or an oligomer of repeating units such as ethylene glycol, amino acid units or amino alkyl groups. The oligonucleotide conjugates of the present invention can be constructed of the following regional elements A-C, A-B-C, A-B-Y-C, A-Y-B-C or A-Y-C. In some embodiments the linker (region Y) is an amino alkyl, such as a C2 - C36 amino alkyl group, including, for example C6 to C12 amino alkyl groups. In a preferred embodiment the linker (region Y) is a C6 amino alkyl group.

Treatment

The term’treatment’ as used herein refers to both treatment of an existing disease (e.g. a disease or disorder as herein referred to), or prevention of a disease, i.e. prophylaxis. It will therefore be recognized that treatment as referred to herein may, in some embodiments, be prophylactic.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to oligonucleotides, such as antisense oligonucleotides, targeting BCL2L11 expression.

The oligonucleotides of the invention targeting BCL2L11 are capable of hybridizing to and inhibiting the expression of a BCL2L11 target nucleic acid in a cell which is expressing the BCL2L11 target nucleic acid.

The BCL2L1 1 target nucleic acid may be a mammalian BCL2L1 1 mRNA or premRNA, such as a human BCL2L11 mRNA or premRNA, for example a premRNA or mRNA originating from the Homo sapiens BCL2 like 11 (BCL2L11 ), RefSeqGene on chromosome 2, exemplified by NCBI Reference Sequence: NG_029006.1 (SEQ ID NO 15). The human BCL2L11 pre-mRNA is encoded on Homo sapiens Chromosome 2, NC_000002.12 (11 1120914.11 1168445, complement). GENE ID = 10018 ( BCL2L11 ).

A mature human mRNA target sequence is illustrated herein by the cDNA sequences SEQ ID NO 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32 or 33.

The oligonucleotides of the invention are capable of inhibiting the expression of BCL2L11 target nucleic acid, such as the BCL2L11 mRNA, in a cell which is expressing the target nucleic acid, such as the BCL2L11 mRNA.

In some embodiments, the oligonucleotides of the invention are capable of inhibiting the expression of BCL2L11 target nucleic acid in a cell which is expressing the target nucleic acid, so to reduce the level of BCL2L11 target nucleic acid (e.g. the mRNA) by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% inhibition compared to the expression level of the BCL2L11 target nucleic acid (e.g. the mRNA) in the cell. Suitably the cell is selected from the group consisting of THP-1 , A431 , MPC-1 1 and J77A.1 cells.

Example 1 provides a suitable assay for evaluating the ability of the oligonucleotides of the invention to inhibit the expression of the target nucleic acid. Suitably the evaluation of a compounds ability to inhibit the expression of the target nucleic acid is performed in vitro, such a gymnotic in vitro assay, for example as according to Example 1.

An aspect of the present invention relates to an antisense oligonucleotide, such as an LNA antisense oligonucleotide gapmer which comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length with at least 90% complementarity, such as is fully

complementary to SEQ ID NO 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32 or 33.

complementary to SEQ ID NO 15, 16, 17, 18, 20, 23, 25, 26, 27, 28, 29, 30, 31 , 32.

In some embodiments, the oligonucleotide comprises a contiguous sequence of 10 - 30 nucleotides, which is at least 90% complementary, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or 100% complementary with a region of the target nucleic acid or a target sequence. In some embodiments, the oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 24, such as 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, or 23, contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 11.

In some embodiments, the oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 24, such as 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, or 23, contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 12.

In some embodiments, the antisense oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 15, such as 13, or 14, 15 contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 13.

In some embodiments, the antisense oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 18, such as 13, 14, 15, 16, or 17, contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 14.

In some embodiments, the antisense oligonucleotide of the invention or the contiguous nucleotide sequence thereof is a gapmer, such as an LNA gapmer, a mixed wing gapmer, or an alternating flank gapmer.

In some embodiments, the antisense oligonucleotide according to the invention, comprises a contiguous nucleotide sequence of at least 10 contiguous nucleotides, such as at least 12 contiguous nucleotides, such as at least 13 contiguous nucleotides, such as at least 14 contiguous nucleotides, such as at least 15 contiguous nucleotides, which is fully

complementary to SEQ ID NO 11.

In some embodiments, the antisense oligonucleotide according to the invention, comprises a contiguous nucleotide sequence of at least 10 contiguous nucleotides, such as at least 12 contiguous nucleotides, such as at least 13 contiguous nucleotides, such as at least 14 contiguous nucleotides, such as at least 15 contiguous nucleotides, which is fully complementary to SEQ ID NO 12.

In some embodiments, the antisense oligonucleotide according to the invention, comprises a contiguous nucleotide sequence of at least 10 contiguous nucleotides, such as at least 12 contiguous nucleotides, such as at least 13 contiguous nucleotides, such as at least 14 contiguous nucleotides, such as at least 15 contiguous nucleotides, which is fully complementary to SEQ ID NO 13.

In some embodiments, the antisense oligonucleotide according to the invention, comprises a contiguous nucleotide sequence of at least 10 contiguous nucleotides, such as at least 12 contiguous nucleotides, such as at least 13 contiguous nucleotides, such as at least 14 contiguous nucleotides, such as at least 15 contiguous nucleotides, which is fully complementary to SEQ ID NO 14.

In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is less than 20 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12 - 24 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12 - 22 nucleotides in length.

In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12 - 20 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12 - 18 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12 - 16 nucleotides in length.

Advantageously, in some embodiments all of the internucleoside linkages between the nucleosides of the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 1 1.

In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 12. In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 13.

In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 14.

In some embodiments, the antisense oligonucleotide is a gapmer oligonucleotide comprising a contiguous nucleotide sequence of formula 5’-F-G-F’-3’, where region F and F’ independently comprise 1 - 8 sugar modified nucleosides, and G is a region between 5 and 16 nucleosides which are capable of recruiting RNaseH.

In some embodiments, the sugar modified nucleosides of region F and F’ are independently selected from the group consisting of 2’-0-alkyl-RNA, 2’-0-methyl-RNA, 2’-alkoxy-RNA, 2’- O-methoxyethyl-RNA, 2’-amino-DNA, 2’-fluoro-DNA, arabino nucleic acid (ANA), 2’-fluoro- ANA and LNA nucleosides.

In some embodiments, region G comprises 5 - 16 contiguous DNA nucleosides.

In some embodiments, wherein the antisense oligonucleotide is a gapmer oligonucleotide, such as an LNA gapmer oligonucleotide.

In some embodiments, the LNA nucleosides are beta-D-oxy LNA nucleosides.

In some embodiments, the internucleoside linkages between the contiguous nucleotide sequence are phosphorothioate internucleoside linkages. Sequence Motifs and Compounds of the Invention

In the compound column, capital letters are beta-D-oxy LNA nucleosides, and LNA C are all 5-methyl C, lower case letters are DNA nucleosides, and a superscript m before a lower case c represent a 5-methyl cytosine DNA nucleoside, and all internucleoside linkages are phosphorothioate internucleoside linkages.

The invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides 12 - 24, such as 12 - 18 in length, nucleosides in length wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence comprising at least 12, such as at least 14, such as at least 15 contiguous nucleotides present in SEQ ID NO 1 , 2, 5, 7 or 10.

The invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides 12 - 24 nucleosides in length, such as 12 - 18 in length, wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence comprising at least 12, such as at least 13, such as at least 14, such as at least 15 contiguous nucleotides present in SEQ ID NO 3, 4, 6, or 9.

The invention provides LNA gapmers according to the invention comprising or consisting of a contiguous nucleotide sequence selected from SEQ ID NO 1 - 10.

The invention provides antisense oligonucleotides selected from the group consisting of: CTCAgaacttacatCA, TCAgaacttacatCAG, GCTacatacgctgGG, ATGctacatacgctGG,

TCAgaacttacatcAGA, CAAatgctacataCGC, CTCAgaacttacatcAG, GAgatagtggttGAAG, TGctacatacgctGGG, GAacttacatcagAAGG; wherein a capital letter is a LNA nucleoside, and a lower case letter is a DNA nucleoside. In some embodiments all internucleoside linkages in contiguous nucleoside sequence are phosphorothioate internucleoside linkages.

Optionally LNA cytosine may be 5-methyl cytosine. Optionally DNA cytosine may be 5- methyl cytosine.

TCAgaacttacatcAGA, CAAatgctacataCGC, CTCAgaacttacatcAG, GAgatagtggttGAAG, TGctacatacgctGGG, GAacttacatcagAAGG; wherein a capital letter is a beta-D-oxy-LNA nucleoside, and a lower case letter is a DNA nucleoside. In some embodiments all internucleoside linkages in contiguous nucleoside sequence are phosphorothioate internucleoside linkages. Optionally LNA cytosine may be 5-methyl cytosine. Optionally DNA cytosine may be 5-methyl cytosine.

TCAgaacttacatcAGA, CAAatgctacataCGC, CTCAgaacttacatcAG, GAgatagtggttGAAG, TGctacatacgctGGG, GAacttacatcagAAGG; wherein a capital letter is a beta-D-oxy-LNA nucleoside, wherein all LNA cytosinese are 5-methyl cytosine, and a lower case letter is a DNA nucleoside, wherein all internucleoside linkages in contiguous nucleoside sequence are phosphorothioate internucleoside linkages, and optionally DNA cytosine may be 5-methyl cytosine.

Method of manufacture

In a further aspect, the invention provides methods for manufacturing the oligonucleotides of the invention comprising reacting nucleotide units and thereby forming covalently linked contiguous nucleotide units comprised in the oligonucleotide. Preferably, the method uses phophoramidite chemistry (see for example Caruthers et al, 1987, Methods in Enzymology vol. 154, pages 287-313). In a further embodiment the method further comprises reacting the contiguous nucleotide sequence with a conjugating moiety (ligand) to covalently attach the conjugate moiety to the oligonucleotide. In a further aspect a method is provided for manufacturing the composition of the invention, comprising mixing the oligonucleotide or conjugated oligonucleotide of the invention with a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

Pharmaceutical Composition

In a further aspect, the invention provides pharmaceutical compositions comprising any of the aforementioned oligonucleotides and/or oligonucleotide conjugates or salts thereof and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS) and pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. In some embodiments the pharmaceutically acceptable diluent is sterile phosphate buffered saline.

In some embodiments the oligonucleotide is used in the pharmaceutically acceptable diluent at a concentration of 50 - 300mM solution.

The compounds according to the present invention may exist in the form of their

pharmaceutically acceptable salts. The term“pharmaceutically acceptable salt” refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of the present invention and are formed from suitable non- toxic organic or inorganic acids or organic or inorganic bases. Acid-addition salts include for example those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethyl ammonium hydroxide. The chemical modification of a pharmaceutical compound into a salt is a technique well known to pharmaceutical chemists in order to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. It is for example described in Bastin, Organic Process Research & Development 2000, 4, 427-435 or in Ansel, In:

Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed. (1995), pp. 196 and 1456-1457. For example, the pharmaceutically acceptable salt of the compounds provided herein may be a sodium salt.

Suitable formulations for use in the present invention are found in Remington's

Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g., Langer (Science 249:1527-1533,

1990). WO 2007/031091 provides further suitable and preferred examples of

pharmaceutically acceptable diluents, carriers and adjuvants (hereby incorporated by reference). Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are also provided in W02007/031091.

Oligonucleotides or oligonucleotide conjugates of the invention may be mixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of

pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11 , more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules. The composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.

In some embodiments, the oligonucleotide or oligonucleotide conjugate of the invention is a prodrug. In particular with respect to oligonucleotide conjugates the conjugate moiety is cleaved of the oligonucleotide once the prodrug is delivered to the site of action, e.g. the target cell.

Applications

The oligonucleotides of the invention may be utilized as research reagents for, for example, diagnostics, therapeutics and prophylaxis.

In research, such oligonucleotides may be used to specifically modulate the synthesis of BCL2L11 protein in cells (e.g. in vitro cell cultures) and experimental animals thereby facilitating functional analysis of the target or an appraisal of its usefulness as a target for therapeutic intervention. Typically the target modulation is achieved by degrading or inhibiting the mRNA producing the protein, thereby prevent protein formation or by degrading or inhibiting a modulator of the gene or mRNA producing the protein.

The present invention provides an in vivo or in vitro method for modulating BCL2L11 expression in a target cell which is expressing BCL2L11, said method comprising administering an oligonucleotide of the invention in an effective amount to said cell.

In some embodiments, the target cell, is a mammalian cell in particular a human cell. The target cell may be an in vitro cell culture or an in vivo cell forming part of a tissue in a mammal.

In diagnostics the oligonucleotides may be used to detect and quantitate BCL2L11 expression in cell and tissues by northern blotting, in-situ hybridisation or similar techniques. For therapeutics, an animal or a human, suspected of having a disease or disorder, which can be treated by modulating the expression of BCL2L11

The invention provides methods for treating or preventing a disease, comprising

administering a therapeutically or prophylactically effective amount of an oligonucleotide, an oligonucleotide conjugate or a pharmaceutical composition of the invention to a subject suffering from or susceptible to the disease.

The invention also relates to an oligonucleotide, a composition or a conjugate as defined herein for use as a medicament. The oligonucleotide, oligonucleotide conjugate or a pharmaceutical composition according to the invention is typically administered in an effective amount.

The invention also provides for the use of the oligonucleotide or oligonucleotide conjugate of the invention as described for the manufacture of a medicament for the treatment of a disorder as referred to herein, or for a method of the treatment of as a disorder as referred to herein.

The disease or disorder, as referred to herein, is associated with expression of BCL2L17. In some embodiments disease or disorder may be associated with a mutation in the BCL2L11 gene. Therefore, in some embodiments, the target nucleic acid is a mutated form of the BCL2L11 sequence.

The methods of the invention are preferably employed for treatment or prophylaxis against diseases caused by abnormal levels and/or activity of BCL2L17.

The invention further relates to use of an oligonucleotide, oligonucleotide conjugate or a pharmaceutical composition as defined herein for the manufacture of a medicament for the treatment of abnormal levels and/or activity of BCL2L 77.

In one embodiment, the invention relates to oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions for use in the treatment of diseases or disorders selected from hepatotoxicity, Alzheimer^'s disease, stroke, epileptic seizures, encephalomyelitis, multiple sclerosis (MS), type 1 diabetes, type 2 diabetes, and graft-vers us-host disease.

Administration

The oligonucleotides or pharmaceutical compositions of the present invention may be administered topical or enteral or parenteral (such as, intravenous, subcutaneous, intra- muscular, intracerebral, intracerebroventricular or intrathecal).

In a preferred embodiment the oligonucleotide or pharmaceutical compositions of the present invention are administered by a parenteral route including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, e.g. intracerebral or intraventricular, intravitreal administration. In one embodiment the active oligonucleotide or oligonucleotide conjugate is administered intravenously. In another embodiment the active oligonucleotide or oligonucleotide conjugate is administered subcutaneously.

In some embodiments, the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is administered at a dose of 0.1 - 15 mg/kg, such as from 0.2 - 10 mg/kg, such as from 0.25 - 5 mg/kg. The administration can be once a week, every 2^nd week, every third week or even once a month. Combination therapies

In some embodiments the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is for use in a combination treatment with another therapeutic agent. The therapeutic agent can for example be the standard of care for the diseases or disorders described above.

The work leading to this invention has received funding from the European Union Seventh Framework Programme [FP7-2007-2013] under grant agreement“HEALTH-F2-2013- 602114" (Athero-B-Cell).

EXAMPLES

Example 1 : Testing in vitro efficacy of antisense oligonucleotides targeting human (and mouse) BCL2L11 mRNA in THP-1 and A431 , J774A.1 and MPC-11 cell lines at single concentration.

THP-1 , A431 , J774A.1 and MPC-1 1 cell lines were purchased from ATCC and maintained as recommended by the supplier in a humidified incubator at 37°C with 5% C02. For assays, 3000 cells/well of A431 or J774A.1 were seeded in a 96 multi well plate in culture media. Cells were incubated for 24 hours before addition of oligonucleotides dissolved in PBS. For suspension cell lines THP-1 and MPC-1 1 , 30.000 cells were seeded in round bottom 96 well plates and oligonucleotides dissolved in PBS added immediately after seeding. Final concentration of oligonucleotides: 25 mM. 3 days after addition of oligonucleotides, the cells were harvested. RNA was extracted using the PureLink Pro 96 RNA Purification kit (Thermo Fisher Scientific) according to the manufacturer’s instructions and eluated in 50mI water. The RNA was subsequently diluted 10 times with DNase/RNase free Water (Gibco) and heated to 90°C for one minute.

For gene expressions analysis, One Step RT-qPCR was performed using qScript™ XLT One-Step RT-qPCR ToughMix®, Low ROX™ (Quantabio) in a duplex set up. The following TaqMan primer assays were used for qPCR: BCL2L1 1 Hs00708019_s1 /(Bcl2H 1

Mm00437796_m1 ) [FAM-MGB] and endogenous control GAPDH, Hs99999905_m1 (Mm99999915_g1 ) [VIC-MGB] All primer sets were purchased from Thermo Fisher Scientific. The relative BCL2L11 mRNA expression level in the table is shown as percent of control (PBS-treated cells).

The BCL2L1 1 mRNA levels from cells treated with a selection of the compounds are shown in figures 1 to 4, evaluated in human THP-1 and A431 cell lines and in mouse MPC-1 1 and J774A.1 cell lines. From the initial library screen we identified 2 motifs on the BCL2L1 1 human transcript which provided surprisingly effective and potent compounds in the cell lines tested: Motif A (SEQ ID NO 1 1 ), and Motif B (SEQ ID NO 12).

Selected Oligonucleotides used:

For Compounds: Capital letters

LNA nucleosides (beta-D-oxy LNA nucleosides were used), all LNA cytosines are 5-methyl cytosine, lower case letters represent DNA nucleosides, DNA cytosines preceded with a superscript ^m represents a 5-methyl C-DNA nucleoside. All internucleoside linkages are phosphorothioate internucleoside linkages.

Example 2: Testing in vitro potency and efficacy of selected oligonucleotides targeting human BCL2L11 mRNA in human THP-1 and A431 cell lines at different concentrations for a dose response curve.

Human A431 and THP-1 cell line was described in Example 1. The assay was performed as described in Example 1 . Concentration of oligonucleotides: from 50 mM, 1 :1 dilutions in 8 steps. 3 days after addition of oligonucleotides, the cells were harvested. RNA extraction and duplex One Step RT-qPCR were performed as described in Example 1 . n=2 biological replicates. IC₅₀ determinations were performed in GraphPad Prism6. The relative BCL2L1 1 mRNA level at treatment with 50 mM oligonucleotide is shown in the table as percent of control (PBS).

The IC50 values for selected oligonucleotides targeting human BCL2L1 1 mRNA in vitro in the human cell lines THP-1 and A431 are shown in Figure 5. The concentration response curves in human cell lines THP-1 and A431 , are provided as Figures 6 and 7 respectively. Example 3: Testing in vitro potency and efficacy of selected oligonucleotides targeting mouse Bcl2H 1 mRNA in mouse MPC-11 and J774A.1 cell lines at different concentrations for a dose response curve.

Mouse MPC-1 1 and J774A.1 cell line was described in Example 1. The assay was performed as described in Example 1. Concentration of oligonucleotides: from 50 mM, 1 :1 dilutions in 8 steps. 3 days after addition of oligonucleotides, the cells were harvested. RNA extraction and duplex One Step RT-qPCR were performed as described in Example 1. n=2 biological replicates. IC50 determinations were performed in GraphPad Prism6. The relative Bcl2H 1 mRNA level at treatment with 50 pM oligonucleotide is shown in the table as percent of control (PBS).

The IC50 values for selected oligonucleotides targeting mouse BCL2L1 1 mRNA in vitro in the mouse cell lines J774A.1 and MPC-1 1 are shown in Figure 8. The concentration response curves in mouse cell lines J774A.1 and MPC-1 1 are provided as Figures 9 and 10, respectively.

Example 4: Mouse in vivo efficacy and tolerance study, 7 days of treatment,

Intravenous IV (tail vein).

Animals

Experiment was performed on female C57BL/6JBom mice. Five animals were included in each group of the study, including a saline control group.

Compounds and dosing procedures

Animals were dosed a single time intravenous (tail vein) with 60mg/kg compound at day 0, and the study was terminated at day 7.

Euthanasia

At the end of the study (day 7) all mice were euthanized with C02 before tissue samples of liver, kidney, mesenteric lymph nodes, heart and spleen tissue were dissected and snap frozen.

Quantification of Bcl2l11 RNA expression Tissue samples were kept frozen until lysed in MagNA Pure LC RNA Isolation Tissue Lysis Buffer (Product No. 03604721001 , Roche) and RNA extraction continued using the MagNA Pure 96 Cellular RNA Large Volume Kit (Product No. 05467535001 , Roche) on a MagNA Pure 96 Instrument (Roche) according to the user’s manual and RNA diluted to 5ng/pl in water.

For gene expressions analysis, One Step RT-qPCR was performed using qScript™ XLT One-Step RT-qPCR ToughMix®, Low ROX™ (Quantabio) in a duplex set up. The following TaqMan primer assays were used for qPCR: Bcl2l1 1 Mm00437796_m1 (FAM-MGB) and endogenous control Gapdh, Mm99999915_g1 (VIC-MGB). All primer sets were purchased from Thermo Fisher Scientific. The relative mRNA expression levels are shown as percent of saline treated control group (Figure 1 1 ).

Claims

1. An antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary to SEQ ID NO 15, wherein the antisense oligonucleotide is capable of inhibiting the expression of human BCL2L11 in a cell which is expressing human BCL2L11 or a pharmaceutically acceptable salt thereof.

2. The antisense oligonucleotide according to claim 1 , wherein the contiguous

nucleotide sequence is fully complementary to a sequence selected from the group consisting of SEQ ID NO 1 1 , SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 14.

3. The antisense oligonucleotide according to claim 1 , wherein the contiguous

nucleotide sequence is fully complementary to SEQ ID NO 11.

4. The antisense oligonucleotide according to claim 1 , wherein the contiguous

nucleotide sequence is fully complementary to SEQ ID NO 12.

5. The antisense oligonucleotide according to claim 1 , wherein the contiguous

nucleotide sequence is fully complementary to a region of SEQ ID NO 15, selected from the group consisting of 306 - 320; 3219 - 3239; 3228 - 3250; 3252 - 3331 ; 3333 - 3352; 3354 - 3384; 3441 - 3465; 3524 - 3557; 3560 - 3596; 3606 - 3624; 3626 - 3640; 3713 - 3736; 3751 - 3773; 3777 - 3868; 3870 - 3955; 3957 - 3971 ; 3995 -

4029; 4043 - 4062; 4064 - 4081 ; 4103 - 4162; 4165 - 4182; 4184 - 4227; 4229 -

4243; 4243 - 4267; 4310 - 4342; 4344 - 4499; 4501 - 4568; 4570 - 4819; 4821 -

4835; 4837 - 4852; 4880 - 4910; 4916 - 4933; 4938 - 4971 ; 4973 - 4989; 4996 -

5023; 5039 - 5086; 5088 - 51 13; 5130 - 5156; 5158 - 5193; 5218 - 5279; 5309 -

5361 ; 5367 - 5426; 5430 - 5448; 5456 - 5501 ; 5523 - 5546; 5548 - 5565; 5567 -

5594; 5596 - 5641 ; 5643 - 5666; 5668 - 5682; 5684 - 5720; 5722 - 5740; 5742 -

5764; 5761 - 5783; 5785 - 5831 ; 5837 - 5886; 5903 - 5920; 5922 - 5937; 5949 -

5965; 5978 - 6010; 6012 - 6031 ; 6037 - 6051 ; 6059 - 6074; 6081 - 6097; 6099 -

6117; 6133 - 6150; 6169 - 6204; 6215 - 6239; 6263 - 6320; 6342 - 6362; 6379 -

6397; 6410 - 6426; 6428 - 6457; 6459 - 6474; 6476 - 6492; 6509 - 6535; 6548 -

6583; 6585 - 6604; 6640 - 6688; 6690 - 6708; 6710 - 6746; 6753 - 6811 ; 6813 -

6836; 6853 - 6909; 6915 - 6952; 6954 - 6994; 7016 - 7037; 7040 - 7060; 7082 -

7096; 7108 - 7142; 7158 - 7173; 7175 - 7223; 7225 - 7242; 7244 - 7298; 7300 -

7317; 7319 - 7397; 7399 - 7452; 7454 - 7487; 7490 - 7518; 7525 - 7550; 7557 -

7576; 7587 - 7601 ; 7625 - 7641 ; 7640 - 7666; 7670 - 7684; 7704 - 7761 ; 7763 - 7816; 7818 - 7871 ; 7893 - 7907; 7919 - 7938; 7940 - 7984; 7986 - 8011; 8013 -

8044; 8040 - 8062; 8052 - 8067; 8069 - 8098; 8100 - 8148; 8150 - 8201; 8215 -

8233; 8249 - 8288; 8297 - 8327; 8352 - 8368; 8372 - 8386; 8391 - 8438; 8442 -

8465; 8469 - 8524; 8526 - 8546; 8549 - 8573; 8603 - 8634; 8670 - 8684; 8727 -

8751; 8753 - 8767; 8773 - 8788; 8836 - 8852; 8854 - 8869; 8885 - 8916; 8918 -

8944; 8975 - 8989; 9002 - 9026; 9028 - 9050; 9065 - 9096; 9099 - 9128; 9130 -

9202; 9219 - 9237; 9226 - 9244; 9241 - 9275; 9288 - 9331 ; 9333 - 9399; 9389 -

9420; 9438 - 9498; 9500 - 9518; 9529 - 9545; 9554 - 9573; 9570 - 9605; 9645 -

9663; 9709 - 9733; 9764 - 9785; 9801 - 9834; 9847 - 9864; 9866 - 9880; 9882 -

9900: 9920 - 9968: 9970 - 9990: 10008 - 10022: 10024 - 10053: 10066 - 10085:

10098 - 10114 10126-10151; 10155-10169 10183-10197 10202 10222 10224 - 10291 10293- 10308; 10306- 10320 10322- 10337 10344- 10369 10371 - 10397 10416- 10477; 10496- 10521 10515- 10530 10523- 10546 10558 - 10580 10576- 10625; 10627- 10702 10705- 10732 10731 - 10748 10750 - 10765 10768- 10783; 10792- 10875 10877- 10908 10917- 10961 10963 - 10977 10970- 10992; 11026- 11041 11055- 11102 11104-11125 11127 - 11153 11159- 11194; 11196-11215 11273- 11290 11310-11372 11395- 11435 11461 - 11475; 11509- 11525 11530- 11545 11547- 11573 11589 - 11606 11609- 11642; 11644- 11672 11695- 11710 11719-11749 11781 - 11821 11876- 11891; 11897- 11919 11921 - 11942 11944- 11974 11987 - 12010 12039- 12068; 12090- 12106 12132-12157 12180-12194 12206 - 12225 12228- 12246; 12242- 12267 12284- 12302 12298- 12315 12345- 12362 12364- 12399; 12413- 12432 12451 - 12483 12517- 12549 12556 - 12595 12597- 12619; 12621 - 12638 12640- 12672 12687- 12710 12712 - 12727 12734- 12748; 12750- 12788 12807- 12836 12866- 12880 12891 - 12915 12925- 12962; 12968- 12992 12994- 13013 13031 - 13064 13078 - 13094 13103- 13123; 13140-13155 13154-13185 13218- 13259 13273 - 13293 13297- 13319; 13361 - 13432 13434- 13452 13465- 13484 13483 - 13533 13546- 13560; 13572- 13596 13607- 13668 13670- 13690 13691 - 13768 13766- 13783; 13821 - 13835 13837- 13863 13882- 13911 13932 - 13952 13953- 13973; 14029- 14067 14069- 14089 14091 - 14134 14156 - 14186 14188- 14203; 14248- 14267 14308- 14324 14326- 14354 14375- 14392 14398- 14454; 14456- 14479 14481 - 14510 14512- 14543 14545- 14586 14597- 14646; 14648- 14669 14694- 14724 14763- 14786 14798 - 14816 14845- 14871; 14887- 14904 14938- 14953 14955- 14980 14991 15008 15010 15032 15053 15078 15081 15095 15126 15151

15172 15200 15225 15255 15257 15288 15290 15310 15312 15356

15359 15401 15403 15417 15430 15462 15465 15500 15502 15525

15540 15558 15568 15606 15620 15659 15661 15701 15703 15728

15730 15771 15781 15807 15809 15866 15881 15904 15906 15947

15959 15975 15993 16029 16031 16082 16084 16098 16100 16122

16124 16143 16157 16172 16174 16206 16210 16234 16243 16269

16283 16309 16322 16336 16353 16381 16390 16406 16437 16470

16515 16529 16537 16567 16579 16624 16632 16679 16758 16801

16812 16847 16859 16892 16894 16908 16910 16958 16973 16994

17038 17072 17079 17099 17127 17156 17160 17184 17186 17221

17238 17254 17256 17303 17310 17341 17354 17385 17396 17419

17432 17448 17460 17480 17482 17496 17518 17536 17538 17555

17557 17588 17593 17610 17612 17627 17637 17663 17665 17683

17710 17741 17743 17760 17787 17834 17839 17881 17887 17923

17925 17943 17962 17998 18000 18014 18018 18032 18034 18070

18072 18091 18098 18115 18117 18142 18139 18179 18182 18207

18204 18224 18256 18299 18301 18345 18347 18364 18366 18397

18396 18418 18430 18459 18468 18482 18484 18513 18515 18536

18538 18567 18603 18617 18633 18652 18688 18755 18755 18778

18794 18836 18832 18864 18880 18906 18914 18938 18933 18948

18940 18963 18973 18987 18989 19007 19039 19062 19071 19097

19099 19130 19152 19184 19189 19207 19221 19255 19257 19299

19313 19327 19325 19350 19366 19391 19423 19470 19473 19528

19574 19624 19626 19647 19649 19673 19675 19707 19720 19770

19797 19812 19814 19831 19833 19892 19924 19980 19982 20001

20003 20044 20069 20084 20095 20140 20142 20186 20204 20228

20230 20262 20270 20296 20308 20322 20332 20395 20404 20471

20503 20532 20543 20578 20580 20601 20603 20625 20653 20672

20674 20703 20705 20763 20772 20792 20798 20861 20863 20893

20884 20942 20944 20964 20984 21002 21004 21056 21058 21072

21106 21 133 21 142 21 159 21 161 21 177 21 187 21217 21218 21237

21239 21281 21290 21307 21308 21341 21343 21364 21378 21402

21407 21435 21437 21462 21501 21530 21547 21565 21567 21594

21596 21613 21615 21635 21649 21694 21699 21722 21724 21774 21785 21833 22324 22339 22334 22348 22362 22381 22404 22426

22427 22463 22463 22477 22479 22515 22515 22542 22544 22570

22572 22599 22604 22620 22634 22665 22667 22688 22702 22723

22726 22740 22750 22805 22807 22828 22851 22868 22880 22913

22960 22990 23028 23092 23107 23122 23143 23182 23192 23210

23243 23268 23276 23309 23319 23402 23408 23432 23434 23458

23460 23483 23485 23506 23529 23548 23600 23621 23664 23678

23680 23699 23701 23757 23759 23804 23831 23857 23859 23878

23916 23974 23972 24009 2401 1 24042 24044 24063 24078 24104

24135 24159 24192 24206 24208 24230 24232 24249 24251 24282

24284 24321 24323 24346 24357 24376 24391 24455 24457 24494

24496 24522 24524 24553 24555 24576 24578 24599 24601 24620

24622 24674 24691 2471 1 24713 24767 24771 24786 24793 24857

24901 24993 25005 25021 25031 25049 25055 25077 25079 25094

25096 25128 25185 25207 25209 25227 25229 25261 25263 25277

25290 25339 25368 25382 25400 25431 25433 25460 25479 25523

25538 25558 25560 25588 25607 25639 25641 25665 25671 25696

25698 25725 25727 25741 25757 25788 25790 25805 25807 25826

25828 25851 25851 25871 25910 25965 25967 26022 26039 26071

26073 26090 26105 26121 26137 26155 26155 26259 26273 26324

26336 26351 26372 26386 26413 26431 26445 26460 26477 26493

26511 26538 26541 26564 26566 26592 26609 26626 26628 26656

26690 26719 26730 26746 26748 26769 26771 26791 26793 2681 1

26813 26852 26869 26894 26906 26924 26930 26973 26987 27010

27012 27028 27044 27075 27078 27095 27097 27119 27146 27160

27179 27199 27201 27217 27219 27233 27243 27259 27261 27284

27290 27316 27326 27345 27365 27408 27410 27437 27486 27520

27537 27562 27620 27646 27649 27670 27675 27712 27714 27736

27753 27796 27798 27832 27852 27869 27871 27902 2791 1 27930

27970 28015 28018 28124 28128 28174 28176 28193 28195 28221

28232 28283 28301 28323 28331 28358 28364 28388 28421 28452

28454 28500 28528 28548 28560 28717 28719 28760 28762 28778

28795 28829 28831 28852 28898 28959 28964 28994 28996 29022

29041 29067 29088 29117 29119 29151 29153 29197 29199 29226

29252 29290 29405 29435 29439 29456 29463 29501 29503 29524 29537 29551 29557 29575 29582 29597 2961 1 29636 29638 29666

29668 29683 29706 29730 29732 29757 29760 29856 29858 29895

29897 29958 29974 30003 30005 30050 30052 30089 30091 30108

301 10 30134 30136 30160 30176 30190 30192 30227 30233 30255

30261 30308 30310 30330 30347 30368 30420 30441 30443 30468

30470 30494 30519 30533 30535 30558 30560 30588 30592 30622

30635 30653 30652 30672 30674 30703 30712 30733 30738 30786

30807 30822 30854 30879 30881 30942 30944 30964 30964 31003

31004 31026 31028 31057 31066 31085 31087 31 116 31 118 31 182

31184 31201 31203 31224 31239 31262 31268 31307 31312 31330

31336 31352 31360 31392 31417 31434 31439 31466 31468 31501

31530 31544 31536 31575 31577 31594 31596 31634 31633 31650

31651 31675 31680 31714 3171 1 31725 31743 31758 31762 31805

31837 31853 31855 31874 31881 31899 31935 31951 31953 31977

31979 32073 32106 32123 32126 32146 32143 32162 32168 32199

32199 32227 32229 32262 32262 32278 32280 32315 32317 32363

32390 32441 32452 32479 32481 32496 32534 32603 32605 32632

32647 32694 3271 1 32750 32797 32826 32828 32861 32870 32885

32906 32926 32935 32952 32979 33002 33004 33037 33057 33084

33086 33109 3311 1 33139 33150 33191 33207 33233 33235 33251

33288 33355 33355 3341 1 33425 33452 33460 33489 33492 33536

33536 33557 33562 33584 33586 33604 33606 33623 33631 33659

33661 33679 33681 33707 33730 33765 33814 33828 33844 33861

33875 33915 33936 33958 33999 34027 34049 34244 34270 34292

34299 34359 34371 34586 34592 34642 34644 34667 34669 34737

34739 34756 34789 34813 34815 34835 34839 34853 34868 34904

34912 34927 34946 34963 34976 35013 35015 35030 35083 35142

35157 35192 3521 1 35234 35253 35307 35340 35370 35392 3541 1

35447 35470 35491 35515 35526 35593 35651 35682 35742 35760

35776 35792 35812 35899 35901 35943 35945 35979 35988 36019

36033 36047 36067 36101 36117 36141 36143 36159 36206 36228

36230 36253 36255 36284 36286 36336 36339 36353 36355 36393

36408 36481 36501 36524 36526 36549 36551 36582 36585 36607

36616 36702 36704 36752 36750 36782 36790 36835 36837 36873

36878 36902 36904 36924 36926 36962 36973 36988 36990 37012 37049 37074 37076 37115 37152 37192 37210 37225 37227 37260

37275 37307 37324 37349 37361 37381 37388 37407 37415 37454

37474 37519 37530 37572 37572 37605 37617 37656 37658 37685

37720 37787 37817 37879 37885 37923 37925 37943 37966 37985

38002 38032 38039 38064 38066 38135 38148 38180 38214 38276

38278 38310 38314 38337 38346 38404 38406 38459 38461 38487

38509 38551 38553 38590 38592 38614 38616 38633 38635 38692

38712 38747 38749 38773 38775 38797 3881 1 38846 38849 38890

38900 38917 38942 38964 38966 38998 39000 39069 39098 39115

391 17 39157 39252 39297 39300 39358 39393 39415 39435 39488

39494 3951 1 39529 39543 39592 39607 39625 39657 39685 39702

39713 39730 39732 39765 39767 39788 39797 39835 39837 39866

39868 39900 39909 39929 39929 39956 39969 40004 40006 40025

40027 40069 40071 40095 40134 40157 40175 40198 40216 40255

40257 40306 40319 40339 40354 40376 40379 40413 40443 40485

40496 40514 40531 40550 40552 40569 40571 40588 40602 40635

40641 40666 40706 40835 40837 40854 40856 40878 40880 40896

40917 40959 40961 40988 40990 41025 41058 41074 41087 41 110

41123 41 149 41 151 41225 41255 41300 41310 41331 41333 41347

41349 41363 41365 41387 41389 41405 41417 41437 41439 41453

41461 41488 41542 41561 41563 41587 41594 41620 41622 41654

41666 41682 41689 41713 41729 41808 41810 41828 41833 41854

41861 41883 41888 41925 41927 41950 41963 41979 41981 42004

42009 42030 42048 42070 42072 42088 42096 42123 42181 42195

42228 42247 42304 42328 42337 42356 42358 42390 42404 42419

42428 42442 42448 42497 42505 42565 42597 42646 42660 42686

42686 42701 42703 42741 42760 42816 42818 42880 42882 42940

42983 43028 43060 43077 43093 43108 43110 43156 43171 43215

43217 43233 43255 43276 43282 43304 43336 43351 43356 43387

43389 43434 43436 43498 43500 43694 43696 43730 43732 43749

43752 43786 43837 43865 43875 43927 43966 43980 43996 44013

44025 44081 44089 4411 1 44156 44182 44201 44218 44220 44255

44269 44290 44316 44341 44355 44389 44391 44417 44419 44448

44450 44473 44481 44507 44508 44528 44530 44545 44547 44597

44636 44653 44665 44697 4471 1 44795 44797 44817 44819 44876 44882 - 44901 44914 - 44943; 44945 - 44963; 44985 - 45010; 45013 - 45057; 45059 - 45097 45099 - 45119; 45121 - 45166; 45176 - 45212; 45229 - 45254; 45256 - 45280 45283 - 45364; 45366 - 45402; 45423 - 45480; 45499 - 45572; 45574 - 45597 45604 - 45622; 45635 - 45662; 45664 - 45687; 45689 - 4571 1 ; 45713 - 45760 45769 - 45809; 45820 - 45873; 45875 - 45959; 45987 - 46031 ; 46052 - 46077 46082 - 46113; 46116 - 46133; 46146 - 46176; 46191 - 46226; 46247 - 46263 46265 - 46280; 46298 - 46325; 46327 - 46344; 46353 - 46383; 46395 - 46410 46441 - 46457; 46482 - 4651 1 ; 46516 - 46533; 46548 - 46573; 46619 - 46675 46688 - 46705; 46717 - 46732; 46739 - 46756; 46758 - 46777; 46779 - 46797 46809 - 46831 ; 46833 - 46875; 46877 - 46917; 46941 - 46958; 46975 - 46990 47007 - 47030; 47032 - 47063; 47065 - 47103; 47105 - 47135; 47143 - 47190 47195 - 47236; 47238 - 47263; 47269 - 4731 1 ; 47313 - 47333; 47335 - 47356 47388 - 47403; 47405 - 47441 ; 47437 - 47456; 47469 - 47509; 47521 - 47572 47570 - 47595; 47597 - 47619; 47632 - 47780; 47820 - 47850; 47852 - 47874 47880 - 48034; 48044 - 48108; 4811 1 - 48133; 48143 - 48159; 48175 - 48191 48193 - 48227; 48245 - 48268; 48282 - 48312; 48316 - 48339; 48369 - 48387 48383 - 48418; 48441 - 48466; 48474 - 48533; 48540 - 48599; 48687 - 48726 48728 - 48760; 48792 - 48827; 48829 - 48860; 48863 - 48903; 48905 - 49023 and 49025 - 49058.

6. The antisense oligonucleotide according to any one of claims 1 - 5, wherein the antisense oligonucleotide is a gapmer oligonucleotide comprising a contiguous nucleotide sequence of formula 5’-F-G-F’-3’, where region F and F’ independently comprise 1 - 8 sugar modified nucleosides, and G is a region between 5 and 16 nucleosides which are capable of recruiting RNaseH.

7. The antisense oligonucleotide according to claim 6, wherein the sugar modified nucleosides of region F and F’ are independently selected from the group consisting of 2’-0-alkyl-RNA, 2’-0-methyl-RNA, 2’-alkoxy-RNA, 2’-0-methoxyethyl-RNA, 2’- amino-DNA, 2’-fluoro-DNA, arabino nucleic acid (ANA), 2’-fluoro-ANA and LNA nucleosides.

8. The antisense oligonucleotide according to claim 6 or 7, wherein region G comprises 5 - 16 contiguous DNA nucleosides.

9. The antisense oligonucleotide according to any one of claims 1 - 8, wherein the antisense oligonucleotide is a LNA gapmer oligonucleotide.

10. The antisense oligonucleotide according to any one of claims 6 - 9, wherein the LNA nucleosides are beta-D-oxy LNA nucleosides.

1 1. The antisense oligonucleotide according to any one of claims 1 - 10, wherein the internucleoside linkages between the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

12. The antisense oligonucleotide according to any one of claims 1 - 11 , wherein the oligonucleotide comprises a contiguous nucleotide sequence selected from the group consisting of: SEQ ID NO 1 , SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 9, and SEQ ID NO 10.

13. The antisense oligonucleotide according to any one of claims 1 - 12, wherein the oligonucleotide comprises or consists of a contiguous nucleotide sequence:

CTCAgaacttacatCA (SEQ ID NO 1 )

TCAgaacttacatCAG (SEQ ID NO 2)

GCTacatacgctgGG (SEQ ID NO 3)

ATGctacatacgctGG (SEQ ID NO 4)

TCAgaacttacatcAGA (SEQ ID NO 5)

CAAatgctacataCGC (SEQ ID NO 6)

CTCAgaacttacatcAG (SEQ ID NO 7)

TGctacatacgctGGG (SEQ ID NO 9)

GAacttacatcagAAGG (SEQ ID NO 10)

wherein a capital letter represents a LNA nucleoside, a lower case letter represents a DNA nucleoside.

14. The antisense oligonucleotide according to any one of claims 1 - 13, wherein the oligonucleotide comprises or consists of a contiguous nucleotide sequence:

CTCAgaacttacatCA (SEQ ID NO 1 )

TCAgaacttacatCAG (SEQ ID NO 2)

GCT acata^mcgctgGG (SEQ ID NO 3)

ATGctacata^mcgctGG (SEQ ID NO 4)

TCAgaacttacatcAGA (SEQ ID NO 5)

CAAatgctacataCGC (SEQ ID NO 6)

CTCAgaacttacatcAG (SEQ ID NO 7)

TGctacata^mcgctGGG (SEQ ID NO 9)

GAacttacatcagAAGG (SEQ ID NO 10)

wherein a capital letter represents a beta-D-oxy LNA nucleoside, a lower case letter represents a DNA nucleoside, wherein each LNA cytosine is 5-methyl cytosine, and mc is 5-methyl cytosine DNA, and wherein the internucleoside linkages between the nucleosides are phosphorothioate internucleoside linkages.

15. A conjugate comprising the oligonucleotide according to any one of claims 1 - 14, and at least one conjugate moiety covalently attached to said oligonucleotide.

16. A pharmaceutical composition comprising the oligonucleotide of claim 1-14 or the conjugate of claim 14 and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.

17. An in vivo or in vitro method for modulating BCL2L11 expression in a target cell which is expressing BCL2L11, said method comprising administering an

oligonucleotide of any one of claims 1-14, the conjugate according to claim 15, or the pharmaceutical composition of claim 16 in an effective amount to said cell.

18. A method for treating or preventing a disease comprising administering a

therapeutically or prophylactically effective amount of an oligonucleotide of any one of claims 1 - 14 or the conjugate according to claim 15 or the pharmaceutical composition of claim 16 to a subject suffering from or susceptible to the disease.

19. The method of claim 18, wherein the disease is selected from the group consisting of hepatotoxicity, Alzheimer^'s disease, stroke, epileptic seizures, encephalomyelitis, multiple sclerosis (MS), type 1 diabetes, type 2 diabetes, and graft-vers us-host disease.

20. The oligonucleotide of any one of claims 1 - 14 or the conjugate according to claim 15 or the pharmaceutical composition of claim 16 for use in medicine.

21. The oligonucleotide of any one of claims 1 - 14 or the conjugate according to claim 15 or the pharmaceutical composition of claim 16 for use in the treatment or prevention of a disease selected from the group consisting of hepatotoxicity, Alzheimer^'s disease, stroke, epileptic seizures, encephalomyelitis, multiple sclerosis (MS), type 1 diabetes, type 2 diabetes, and graft-versus-host disease.

22. Use of the oligonucleotide of claim 1 - 14 or the conjugate according to claim 15 or the pharmaceutical composition of claim 16, for the preparation of a medicament for treatment or prevention of a disease selected from the group consisting of hepatotoxicity, Alzheimer^'s disease, stroke, epileptic seizures, encephalomyelitis, multiple sclerosis (MS), type 1 diabetes, type 2 diabetes, and graft-versus-host disease.